Peptides that block the binding of IgG to FcRn

ABSTRACT

The invention relates to peptides which bind to human FcRn and inhibit binding of the Fc portion of an IgG to an FcRn, thereby modulating serum IgG levels. The disclosed compositions and methods may be used for example, in treating autoimmune diseases and inflammatory disorders. The invention also relates to methods of using and methods of making the peptides of the invention.

PRIOR APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.60/774,853, filed Feb. 17, 2006, and 60/805,634, filed Jun. 23, 2006,the contents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention generally relates to the field of immuno-modulators. Morespecifically, the invention relates to peptides which bind to the Fcneonatal Receptor (FcRn) and inhibit binding of the FcRn to a Fc portionof an immunoglobulin G (IgG), thereby modulating serum IgG levels. Theinvention further relates to peptide modulators of FcRn activity thatcan prevent FcRn from functioning in cellular mechanisms related to themaintenance of IgG levels in the serum, medicaments comprising thosepeptides, and methods of treating a subject for diseases and disordersthat can be alleviated by lowering serum IgG levels by administeringthose medicaments.

BACKGROUND OF THE INVENTION

The most abundant antibody isotype in the serum is IgG, which has acritical role in mediating protection against pathogens as well as inmediating allergic and inflammatory responses that hasten recruitment ofimmune system components to the tissues, mucosae, and dermal surfaces.Junghans, Immunologic Research 16 (1):29 (1997). Moreover, IgG is also akey component of a variety of autoimmune diseases.

Under normal conditions, the half-life of IgG in the serum is aprolonged period relative to the serum half-life of other plasmaproteins. For example, the serum half-life of IgG is 5 to 7 days in miceand 22-23 days in humans. Roopenian et al., J. Immunology 170:3528(2003); Junghans and Anderson, Proc. Natl. Acad. Sci. USA 93:5512(1996). In part, this long half-life of IgG is due to its binding to theFc receptor, FcRn. FcRn binds to the constant region of IgG, known asFc. Although FcRn was originally characterized as a neonatal transportreceptor for maternal IgG, it also functions in adults to protect IgGfrom degradation. FcRn binds to pinocytosed IgG to protect it fromdegradative lysosomes and then recycles it back to the extracellularcompartment. Junghans and Anderson, Proc. Natl. Acad. Sci. USA 93:5512(1996), Roopenian et al., J. Immunology 170:3528 (2003). If theconcentration of IgG reaches a level that exceeds available FcRn,unbound IgG will not be protected from degradative mechanisms and willconsequently have a shorter serum half-life. Brambell et al., Nature203:1352 (1964). Furthermore, although FcRn is expressed on the cellsurface, it is believed that much of FcRn is intracellular and isassociated with endoplasmic vesicle membranes, and that the interactionbetween IgG and FcRn occurs intracellularly after IgG is pinocytosedinto the cell.

Structurally, FcRn exists as a heterodimer composed of one light chain,termed a beta (β) chain, and one non-covalently bound heavy chain,termed an alpha (α) chain. The light chain of FcRn, which is betterknown as β₂-microglobulin (β₂m), is also a component of the MajorHistocompatibility complex I (MHC I). The FcRn a chain is a 46 kDprotein composed of an extracellular domain that is divided into threesubdomains, α1, α2, and α3; a transmembrane region; and a relativelyshort cytoplasmic tail. Burmeister et al., Nature 372:336 (1994).

FcRn was first identified in the neonatal rat gut where it functions tomediate the absorption of IgG antibody from the mother's milk andfacilitates its transport to the circulatory system. Leach et al., J.Immunology 157:3317 (1996). FcRn has also been isolated from the humanplacenta where it mediates absorption and transport of maternal IgG tothe fetal circulation. In adults, FcRn is expressed in epithelial tissue(U.S. Pat. Nos. 6,030,613 and 6,086,875), such as, but not limited to,the lung (Israel et al., Immunology 92:69 (1997)), intestinal and renalproximal tubular epithelium (Kobayashi et al., Am. J. Physiol. (2002);Renal Physiol. 282:F358 (2002)), as well as nasal, vaginal, and biliarytree surfaces. In addition, the ubiquitous expression of FcRn onendothelial cells is suggestive of its importance in IgG homeostasis.Ward et al., International Immunology 15 (2):187 (2002); Ghetie et al.,Eur. J. Immunology 26:690 (1996).

In general, FcRn functions in IgG homeostasis by antagonizing thecatabolism of IgG by binding to the Fc portion of IgG. Once pinocytosed,IgG is captured in intracellular vacuoles that are beginning to fusewith acidic early endosomes. Crystallographic studies suggest thestoichiometry of the FcRn-IgG complex is composed of two molecules ofFcRn to one IgG (Burmeister et al., Nature 372:336 (1994)) and bindingof the two molecules is thought to occur on the Fc portion of IgG nearthe interface of the CH2 and CH3 domains (Burmeister et al., Nature372:379 (1994)). The endosomal fusion event represents one stage of thelysosomal degradative pathway, which degrades or catabolizes complexbiomolecules contained within the endosome into constitutive components.The low pH environment of the early endosome promotes the binding ofFcRn to IgG as well as the release of any antigen bound to the IgG.Consequently, the antigen is degraded and the FcRn-IgG complex avoidsdegradation and is ultimately recycled to the cell surface where thephysiological pH of the extracellular environment promotes the releaseof the IgG from FcRn.

In order to study the contributions of FcRn to IgG homeostasis, micehave been engineered so that at least part of the genes encoding β₂m andFcRn heavy chain have been “knocked out” so that these proteins are notexpressed. WO 02/43658; Junghans and Anderson, Proc. Natl. Acad. Sci.USA 93:5512 (1996). In both of these knockout mouse lines, the half-lifeand the concentration of IgG in the serum are dramatically reduced,suggesting a FcRn-dependent mechanism related to IgG homeostasis.

The inhibition of IgG binding to FcRn reduces IgG serum half-life bypreventing IgG recycling. Therefore, agents that block or antagonize thebinding of IgG to FcRn may be used in methods of regulating, treating orpreventing disorders involving immune reactions, such as, e.g.,autoimmune and inflammatory diseases and disorders characterized by thepresence of inappropriately expressed IgG antibodies. One example of amethod of blocking IgG Fc binding to FcRn involves the generation ofblocking antibodies to FcRn. Indeed, antibodies capable of blocking thebinding of FcRn with IgG have been generated using a FcRn heavy chainknockout mouse line (WO 02/43658). Recently, peptides have beenidentified that bind to FcRn complexes. Kolonin et al., Proc. Natl.Acad. Sci. USA 99(20):13055-60 (2002); U.S. Pat. No. 6,212,022. However,at this time additional agents are needed to regulate, treat, or preventconditions, diseases, and disorders characterized by immune reactions.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides peptides which specificallybind to FcRn and inhibit IgG Fc from binding to FcRn, thereby preventingIgG from recycling by preventing FcRn from functioning in its role ofprotecting IgG from degradation by the lysosomes. In exemplaryembodiments, the peptides bind to FcRn and inhibit the IgG1, IgG2, IgG3,or IgG4 subclasses of Fc from binding to FcRn.

In some embodiments, the peptides of the invention comprise thesequence:-Gly-X₆—X₇—X₈—X₉—X₁₀—X₁₁—

wherein:

-   -   X₆ is chosen from positively charged amino acids, aromatic amino        acids, positively charged aromatic amino acids, and analogs        thereof;    -   X₇ is chosen from phenylalanine and phenylalanine analogs,    -   X₈ and X₉ are each independently chosen from glycine, sarcosine,        aspartic acid, D-amino acids, α-aminoisobutyric acid, and        analogs thereof, or X₈, when taken together with X₉, forms a        dipeptide mimetic;    -   X₁₀ is chosen from amino acids and analogs thereof, or X₁₀, when        taken together with X₉, forms a dipeptide mimetic;    -   X₁₁ is chosen from tyrosine and tyrosine analogs.

Alternatively, the peptides of the invention may comprise the sequence:R₁-Gly-X₆—X₇—X₈—X₉—X₁₀—X₁₁—R₂

wherein:

-   -   R₁ has the formula X₁—X₂—X₃—X₄—        -   wherein            -   X₁ is chosen from hydrogen, acyl, and amino acid                protecting groups;            -   X₂ is absent or is chosen from an amino acid and                peptides of 2-15 amino acids in length, and analogs                thereof;            -   X₃ is absent or is an amino acid or analog thereof that                is capable of forming a bridge with X₁₀, X₁₂, or X₁₃,                wherein the bridge is chosen from an amino terminus to                carboxy terminus bridge, a side chain to backbone                bridge, and a side chain to side chain bridge; and            -   X₄ is absent or is chosen from an amino acid, peptides                of 2-15 amino acids in length, and analogs thereof;    -   X₆, —X₇, —X₈, —X₉, —X₁₀, and —X₁₁ are as defined above, and    -   R₂ has the formula —X₁₂—X₁₃—X₁₄—X₁₅        -   wherein            -   X₁₂ is absent or is an amino acid or analog thereof;            -   X₁₃ is absent or is an amino acid or analog thereof;            -   X₁₄ is absent or is chosen from an amino acid, peptides                of 2-15 amino acids in length, and analogs thereof; and            -   X₁₅ is an amino group or a carboxy protecting group.

The peptides of the invention are typically at least 7 and as many as 50amino acids long. Peptides of the invention may exist as a multimer,such as, e.g., a dimer, a trimer, or a tetramer. In some embodiments,the peptides of the invention may be more susceptible to pinocytosis,which enables more rapid binding of the peptide and consequently, lessexcretion by the kidney. The invention further relates to pharmaceuticalcompositions comprising one or more peptides of the invention.

Peptides of the invention may comprise:

a) an amino acid sequence chosen from:

QRFCTGHFGGLYPCNGP, (SEQ ID NO:1) GGGCVTGHFGGIYCNYQ, (SEQ ID NO:2)KIICSPGHFGGMYCQGK, (SEQ ID NO:3) PSYCIEGHIDGIYCFNA, (SEQ ID NO:4) andNSFCRGRPGHFGGCYLF; (SEQ ID NO:5)

b) an amino acid sequence that is substantially identically to one ormore of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ IDNO:5;

c) an amino acid sequence that is at least 80% identical to one or moreof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5;

d) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by one, two, three, four, or five deletion, substitution, oraddition mutations;

e) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by at least one amino acid substitution, wherein the at leastone amino acid is substituted with a naturally occurring amino acid;

f) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by at least one amino acid substitution, wherein the at leastone amino acid is substituted with a D-amino acid and analogs thereof;

g) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by at least one amino acid substitution, wherein the at leastone amino acid is substituted with an N-methylated amino acid;

h) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by at least one amino acid substitution, wherein the at leastone amino acid is substituted with a non-naturally occurring amino acid;and

i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5modified by at least one amino acid substitution, wherein the at leastone amino acid is substituted with an amino acid mimetic.

The invention further relates to a method of regulating IgG levels inthe serum of a subject comprising administering to the subject atherapeutically effective amount of a composition comprising one or morepeptides of the invention capable of binding to and preventing the FcRnfrom binding to the Fc portion of an IgG molecule. In certainembodiments, the methods of the invention are employed to reduce thehalf-life of soluble IgG in the serum of a subject. The result ofadministering a composition of the invention is that the half-life ofsoluble IgG in the serum of the subject is reduced compared to thehalf-life of IgG in the serum of the subject prior to administration ofthe peptide.

The invention further provides methods for inhibiting binding of the Fcportion of a human IgG to FcRn to effect a decrease in the serumconcentration of the Fc portion of IgG for a FcRn as compared to theserum concentration of IgG before treatment. The method of decreasingserum concentration of IgG comprises administering to the subject atherapeutically effective amount of a composition comprising one or morepeptides of the invention capable of binding to FcRn and preventing theFcRn from binding to the Fc portion of an IgG molecule. In oneembodiment, the decrease in the serum concentration of human IgG is atleast 5%, such as a decrease of at least 15%, or a decrease in the serumconcentration of human IgG of at least 25%.

One embodiment of the invention provides a method of treating a subjectwho has at least one autoimmune disease comprising administering to thesubject a therapeutically effective amount of a composition comprisingone or more peptides of the invention capable of binding to FcRn andpreventing the FcRn from binding to the Fc portion of an IgG molecule.An alternate embodiment of the invention provides a method of treating asubject with at least one inflammatory disorder by administering to thesubject a therapeutically effective amount of a composition comprisingone or more peptides of the invention capable of binding to FcRn andpreventing the FcRn from binding to the Fc portion of an IgG molecule.In other embodiments, the methods of the invention may be used toprevent, treat, or regulate an immune response to a therapeutic proteinor a gene therapy vector.

Additional embodiments, objects, and advantages of the invention are setforth in part in the description which follows and in part, will beobvious from the description, or may be learned by practice of theinvention. These embodiments, objects, and advantages of the inventionmay be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are only exemplary and explanatoryand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cDNA sequences of the human full length FcRn and humanbeta-2 microglobulin (β₂m) open reading frames (ORFs).

FIG. 2 shows an overview of the synthesis of N-terminal aldehyde peptidemonomers (SEQ ID NO: 319).

FIG. 3 shows an overview of the synthesis of peptide dimers by reductivealkylation. The synthesis of Peptide No. 270 is shown as an illustrativeexample.

FIG. 4 shows an overview of the synthesis of peptide dimers by using abis-thiol linker containing peptide and a bromoacetylated peptide. Thesynthesis of Peptide No. 100 is shown as an illustrative example.Horizontal brackets placed above the peptide sequence indicate thepresence of a bridge (SEQ ID NO: 320).

FIG. 5 shows the synthesis of peptide dimers using a thiollinker-containing peptide and a bromoacetylated peptide. The synthesisof Peptide No. 122 is shown as an illustrative example. Horizontalbrackets placed above the peptide sequence indicate the presence of abridge (SEQ ID NO: 320).

FIG. 6 shows the synthesis of peptide dimers using a diacid containinglinker. The synthesis of Peptide No. 283 is shown as an illustrativeexample. Horizontal brackets placed below the peptide sequence indicatethe presence of a bridge (SEQ ID NO: 321).

FIG. 7 shows the synthesis of peptide dimers using an amine containinglinker. The synthesis of Peptide No. 280 is shown as an illustrativeexample. Horizontal brackets placed below the peptide sequence indicatethe presence of a bridge (SEQ ID NO: 321).

FIG. 8 shows the synthesis of peptide-Fc fusions using a peptidealdehyde and the protein CysFc. The synthesis of Peptide No. 252-Fc isshown as an illustrative example. Horizontal brackets placed below thepeptide sequence indicate the presence of a bridge.

FIG. 9 shows the kinetics of human IgG catabolism in TG32B mice (miceengineered to express the human FcRn and human β₂m, but not the murineFcRn or β₂m).

FIG. 10 shows that the catabolism kinetics of biotinylated human IgG incynomolgous monkeys is accelerated following the intravenous injectionof Peptide No. 270.

FIG. 11 shows the serum levels of endogenous IgG in cynomolgous monkeysfollowing the intravenous injection of Peptide No. 270.

FIG. 12 is a representation of the results shown in FIG. 11, wherein thelevels of endogenous cynomologous monkey IgG have been normalized to T₀.levels.

FIG. 13 shows the levels of endogenous serum albumin in cynomolgousmonkeys following the intravenous injection of Peptide No. 270.

FIG. 14 shows the levels of endogenous IgM in cynomolgous monkeysfollowing the intravenous injection of Peptide ID No. 270.

FIG. 15 shows the kinetics of human IgG catabolism in TG32B micefollowing the intravenous injection of Peptide No. 231, Peptide No. 274,and Peptide No. 252-Fc.

FIG. 16 shows the kinetics of human IgG catabolism in TG32B micefollowing the intravenous injection of Peptide No. 270.

FIG. 17 shows the kinetics of human IgG catabolism in TG32B micefollowing intravenous injection of Peptide No. 283.

FIG. 18 shows the molecular weight of Peptide No. 289 by SDS-PAGEanalysis of purified Peptide No. 289 on a 4-20% Tris-Gly gel. Lane 1contains molecular weight markers. Lane 2 contains unconjugatedPEG_(30kDa) starting material. Lane 3 contains crude reaction mixture.Lane 4 contains purified Peptide No. 289.

FIG. 19 shows the kinetics of human IgG catabolism in TG32B micefollowing intravenous injection of Peptide No. 289.

FIG. 20 shows the kinetics of biotinylated human IgG catabolism incynomolgus monkeys following intravenous injection of 5 mg/kg PeptideNo. 283.

FIG. 21 shows the kinetics of biotinylated human IgG catabolism incynomolgus monkeys following intravenous injection of 1 mg/kg PeptideNo. 283.

FIG. 22 shows the effect of Peptide No. 283 (5 mg/kg) on theconcentration of IgG in cynomolgus monkeys.

FIG. 23 shows the effect of Peptide No. 283 (1 mg/kg) on theconcentration of IgG in cynomolgus monkeys.

FIG. 24 shows the effect of Peptide No. 283 (5 mg/kg, 3×/week, iv) onthe concentration of albumin in cynomolgus monkeys.

DESCRIPTION OF THE EMBODIMENTS I. Definitions

“Affinity” refers to the characteristics of the binding interactionbetween two biologically active molecules that indicates the strength ofthe binding interaction. The measure of affinity is reported as adissociation constant (K_(D)), which is the concentration, normallyreported in nM, pM, or fM, in a solution comprising a biologicallyactive molecule at which the biologically active molecule begins to nolonger bind (dissociate) to its binding partner under specifiedconditions. Affinity strength is inversely related to the value of theK_(D).

The term “amino acid,” as used herein, refers to a compound containing acarboxylic acid group and an amino group. For example, an amino acid mayhave the structural formula H₂N—[C(R)(R′)]_(n)—C(O)OH, where n is aninteger greater than or equal to one, and R and R′ are independentlyselected from hydrogen and amino acid side chains, and where R and R′may be taken together to form a carbocyclic or heterocyclic ring. Forexample, when n is equal to one, the amino acid of the formulaH₂N—[C(R)(R′)]—C(O)OH is an alpha amino acid, and when n is equal totwo, the amino acid of the formula H₂N—C(R₁)(R₁′)—C(R₂)(R₂′)—C(O)OH is abeta amino acid, where R₁, R₁′, R₂, and R₂′ are each independentlychosen from amino acid side chains, and where R and R′, or R₂, and R₂′,may be taken together to form a carbocyclic or heterocyclic ring. Theterm “amino acid residue,” as used herein, refers to an amino acid thatis part of a peptide or protein, and having the formula—N(H)—[C(R)(R′)]_(n)—C(O)—. The term “amino acid side chain” as usedherein, refers to any side chain from a naturally-occurring or syntheticamino acid. For example, methyl may be referred to as an alanine sidechain, and 2-amino-1-ethyl may be referred to as the side chain of2,4-diaminobutanoic acid.

An amino acid may have R or S chirality at any chiral atom. The Fischerconvention is used to designate the chirality of the alpha amino carbonof alpha amino acids, if the alpha amino carbon is chiral, as L- or D-.A “D-amino acid” is an amino acid having a D configuration at the alphacarbon. Where no specific configuration is indicated, one skilled in theart would understand the amino acid to be an L-amino acid. The aminoacids described herein may also be in the form of racemic, non-racemic,and diastereomeric mixtures.

Exemplary amino acids may be chosen from the twenty encoded amino acidsand analogs thereof, as well as from, e.g., other α-amino acids, β-aminoacids, γ-amino acids, δ-amino acids, and ω-amino acids. Non-encodedamino acids are well known in the peptide art, such as those describedin M. Bodanszky, Principles of Peptide Synthesis, 1st and 2nd reviseded., Springer-Verlag, New York, N.Y., (1984) and (1993), and Stewart andYoung, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.,Rockford, Ill., (1984). Encoded and non-encoded amino acids and aminoacid analogs can be purchased commercially from, e.g., Novabiochem;Bachem; Sigma Chemical Co.; Advanced Chemtech, or synthesized usingmethods known in the art.

An amino acid may be chosen from, e.g., alanine, β-alanine,α-aminoadipic acid, 2-aminobutanoic acid, 4-aminobutanoic acid,1-aminocyclopentanecarboxylic acid, 6-aminohexanoic acid,2-aminoheptanedioic acid, 7-aminoheptanoic acid, 2-aminoisobutyric acid,aminomethylpyrrole carboxylic acid, 8-amino-3,6-dioxa-octanoic acid,aminopiperidinecarboxylic acid, 3-amino-propionic acid, aminoserine,aminotetrahydropyran-4-carboxylic acid, arginine, asparagine, asparticacid, azetidine carboxylic acid, benzothiazolylalanine, butylglycine,carnitine, 4-chlorophenylalanine, citrulline, cyclohexylalanine,cyclohexylstatine, cysteine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid, dihydroxyphenylalanine, dimethylthiazolidinecarboxylic acid, glutamic acid, glutamine, glycine, histidine,homoserine, hydroxyproline, isoleucine, isonipecotic acid, leucine,lysine, methanoproline, methionine, norleucine, norvaline, ornithine,p-aminobenzoic acid, penicillamine, phenylalanine, phenylglycine,piperidinylalanine, piperidinylglycine, proline, pyrrolidinylalanine,sarcosine, selenocysteine, serine, statine, tetrahydropyranglycine,thienylalanine, threonine, tryptophan, tyrosine, valine,allo-isoleucine, allo-threonine, 2,6-diamino-4-hexanoic acid,2,6-diaminopimelic acid, 2,3-diaminopropionic acid, dicarboxidine,homoarginine, homocitrulline, homocysteine, homocystine,homophenylalanine, homoproline, and 4-hydrazinobenzoic acid.

Amino acids are usually classified by properties of the side chain intofour groups: acidic, basic, hydrophilic (polar), and hydrophobic(nonpolar). Amino acids described herein may be identified by their fullnames or by the corresponding standard one- or three-letter codes. Lowercase single-letter codes are used to indicate D-chirality.

An “amino acid analog” is an amino acid, or a small molecule mimetic ofan amino acid, that shares a common chemical, charge, steric, or otherproperty of a given amino acid. For example, analogs of alanine include,e.g., β-alanine, ethylglycine, α-aminoisobutryic acid, and D-alanine;analogs of cysteine include, e.g., homocysteine, D-cysteine, andpenicillamine; analogs of phenylalanine include, e.g.,3-fluorophenylalanine, 4-methylphenylalanine, phenylglycine,1-naphthylalanine, and 3,3-diphenylalanine, 4-aminophenylalanine,pentafluorophenylalanine, 2-pyridylalanine, 3-pyridylalanine,4-nitrophenylalanine, 2-pyrrolidinylalanine, 3-piperidylalanine,4-piperidylalanine; and analogs of histidine include, e.g.,1-methylhistidine, 2,4-diaminobutyric acid, thiazolylalanine,2,3-diaminopropionic acid, guanylalanine, 2-pyridylalanine,3-pyridylalanine, 4-pyridylalanine, thienylalanine, ornithine,4-guanylphenylalanine, and 4-aminophenylalanine.

“Amino protecting group,” as used herein, refers to any substituent thatmay be used to prevent an amino group on a molecule from undergoing achemical reaction while chemical change occurs elsewhere in themolecule. An amino protecting group can be removed under the appropriatechemical conditions. Numerous amino protecting groups are known to thoseskilled in the art, and examples of amino protecting groups, methods fortheir addition, and methods for their removal can be found in T. W.Green, Protective Groups in Organic Synthesis, John Wiley and Sons, NewYork, 1991; Chapter 7, M. Bodanszky, Principles of Peptide Synthesis, 1st and 2nd revised ed., Springer-Verlag, New York, N.Y. (1984) and(1993), and Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed.,Pierce Chemical Co., Rockford, Ill. (1984), the disclosures of which areincorporated herein by reference. The term “protected(monosubstituted)amino” means there is an amino-protecting group on the(monosubstituted)amino nitrogen atom. In addition, the term “protectedcarboxamide” means there is an amino-protecting group on the carboxamidenitrogen. Examples of such amino-protecting groups include the formyl(“For”) group, the trityl group, the phthalimido group, thetrichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetylgroups, urethane-type blocking groups, such as t-butoxycarbonyl (“Boc”),2-(4-biphenylyl)propyl-2-oxycarbonyl (“Bpoc”),2-phenylpropyl-2-oxycarbonyl (“Poc”), 2-(4-xenyl)isopropoxycarbonyl,1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl,2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl (“Ddz”),2-(p-toluoyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluoylsulfonyl)-ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphino)-ethoxycarbonyl, 9-fluorenylmethoxycarbonyl(“Fmoc”), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-phenyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyl-oxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, isobornyloxycarbonyl,1-piperidyloxycarbonyl, benzyloxycarbonyl (“Cbz”),4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,α-2,4,5-tetramethylbenzyloxycarbonyl (“Tmz”),4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxy-carbonyl, 4-cyanobenzyloxycarbonyl,4-(decyloxy)benzyloxycarbonyl and the like; the benzoylmethylsulfonylgroup, dithiasuccinoyl (“Dts”), the 2-(nitro)phenylsulfenyl group(“Nps”), the diphenyl-phosphine oxide group and like amino-protectinggroups. For example, amino protecting groups may be chosen from Boc,Cbz, and Fmoc. More than one variety of amino protecting group may beemployed so long as the derivatized amino group is stable to theconditions of the subsequent reaction(s) and can be removed at theappropriate point without disrupting the remainder of the compounds.

The term “aromatic,” as used herein, refers to a mono-, bi-, or othermulti-carbocyclic, aromatic ring system. The aromatic group mayoptionally be fused to one or more rings chosen from aromatics,cycloalkyls, and heterocyclyls. Aromatics can have from 5-14 ringmembers, such as, e.g., from 5-10 ring members. One or more hydrogenatoms may also be replaced by a substituent group selected from acyl,acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aromatic,aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxyamido,carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino,halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstitutedamino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio,thioacylamino, thioureido, and ureido. Nonlimiting examples of aromaticgroups include phenyl, naphthyl, indolyl, biphenyl, and anthracenyl.

An “aromatic amino acid,” as used herein, is an amino acid having a sidechain that comprises an aromatic ring structure. Aromatic amino acidsinclude, for example, histidine, tyrosine, tryptophan, phenylalanine,1-naphthylalanine, and 4-pyridylalanine.

“Carboxy-protecting group” refers to one of the ester derivatives of thecarboxylic acid group commonly employed to block or protect thecarboxylic acid group while reactions are carried out on otherfunctional groups on the compound. Examples of such carboxylic acidprotecting groups include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl,benzhydryl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl,2-phenylpropyl, trimethylsilyl, t-butyldimethylsilyl, phenacyl,2,2,2-trichloroethyl, β-(trimethylsilyl)ethyl,.beta.-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)-propenyl and like moieties. The species ofcarboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the conditions of subsequentreaction(s) and can be removed at the appropriate point withoutdisrupting the remainder of the molecule. Further examples of thesegroups are found in E. Haslam, Protective Groups in Organic Chemistry,J. G. W. McOmie, Ed., Plenum Press, New York, N.Y. (1973), Chapter 5,and T. W. Greene, Protective Groups in Organic Synthesis, 2nd ed., JohnWiley and Sons, New York, N.Y. (1991), Chapter 5. A related term is“protected carboxy,” which refers to a carboxy group substituted withone of the above-described carboxy-protecting groups.

“Bind,” “binding,” and “bound” refer to a noncovalent interactionbetween a polypeptide and another biologically active molecule that isdependent on the steric and chemical complementarity of the twomolecules. The steric and chemical complementarity of polypeptides isdetermined by a specific amino acid sequence.

“Biologically active molecules” refer to peptides, nucleic acids, and/orsmall molecules, such as small organic or inorganic molecules as well asfragments thereof, capable of treating a disease or condition byperforming a function or an action, or stimulating or responding to afunction, an action or a reaction, in a biological context (e.g. in anorganism, a cell, or an in vitro model thereof).

A “bridge” refers to a covalent bond between two non-adjacent aminoacids, amino acid analogs, or other chemical moieties in a peptide. Thebridge may be, e.g., a backbone to backbone, side chain to backbone, orside chain to side chain bridge. The bridge may be prepared by acyclization reaction that results in the formation of a new amide,ester, ether, thioether, alkene, or disulfide bond. A backbone tobackbone bridge, for example, results from the formation of a lactam atthe N- and C-termini.

A “cyclic peptide” refers to a peptide having an intramolecular bondbridging two non-adjacent amino acids.

A peptide “dimer,” as used herein, is a molecule comprising a first andsecond peptide chain that may be the same or different. The dimer mayfurther comprise at least one optional linker to which the two peptidesare covalently bound.

A “dipeptide mimetic” is substantially similar (e.g., substantiallyisosteric, or having a substantially similar position or orientation) toa dipeptide such as, for example, glycylglycine. The dipeptide mimeticmay comprise any combination of linked molecules as long as thestructural limitations listed above, are recognized. Dipeptide mimeticsmay have one or more peptide linkages optionally replaced by a linkageselected from, for example: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans) —COCH₂—CH(OH)CH₂—, and —CH₂ SO—, by methods well known in theart. The skilled artisan will recognize that dipeptide mimetics alsoinclude, for example, β-turn mimetics. See, e.g., R. M. Friedinger, J.Med. Chem. 46:5553-5566 (2003), and S. Hanessian, Tetrahedron53:12789-12854 (1997).

Nonlimiting examples of dipeptide mimetics include:

(D,L-Friedinger's lactam);

(L,L-Friedinger's lactam);

-   (3S)-3-amino-2-oxo-1-piperidine-acetic acid and    (3R)-3-amino-2-oxo-1-piperidine-acetic acid;-   (3S)-3-amino-2-oxo-1-azepine acetic acid and    (3R)-3-amino-2-oxo-1-azepine acetic acid;-   (3S)-3-amino-2-oxo-1-pyrrolidine acetic acid and    (3R)-3-amino-2-oxo-1-pyrrolidine acetic acid;-   (3R)-3-amino-1-carboxymethyl-valerolactam; and-   3-amino-N-1-carboxymethyl-2,3,4,5-tetrahydro-1H-[1]-benzazepine-2-one.

The term “disulfide bridge” refers to the covalent bond formed betweenthe sulfhydryl (i.e., thiol or mercaptan) groups of 2 amino acids (suchas, e.g., cysteine, penicillamine, and homocysteine) upon oxidation. Adisulfide bridge may bridge two amino acids contained in the same linearpeptide, resulting in the cyclization of the linear peptide. A disulfidebridge may also be formed between two peptides, thereby producing apeptide dimer.

A “domain” is a region of a peptide or peptides having some distinctivephysical feature or role including, for example, an independently foldedstructure composed of one section of a peptide chain. A domain may bindto another domain that is the same or different. A domain may containthe sequence of the distinctive physical feature of the peptide or itmay contain a fragment of the physical feature which retains its bindingcharacteristics (i.e., it can bind to a second domain).

“Effective dose,” “effective amount,” “therapeutically effectiveamount,” and the like, refer to an amount of an agent sufficient toprovide a desired physiological, pharmacological, and/or cognitivechange that may vary depending on the patient, the disease, and thetreatment. The amount may either be a dose for the treatment of asubject believed to have a particular disorder, in which case it shouldsufficiently alleviate or ameliorate the symptoms of the disorder orcondition, or be a prophylacetic dose, in which case it should besufficient to prevent, partially or completely, the appearance ofsymptoms in the subject.

The term “fusion,” as used herein, refers to a covalent conjugatebetween a peptide of the invention and another molecule, which may be,e.g., a protein, a peptide, a small molecule, a polymer (e.g.,polyethylene glycol or a polysaccharide), or a nucleic acid. Apeptide-small molecule or peptide-polymer fusion may be preparedsynthetically, whereas a peptide-protein or peptide-peptide fusion maybe prepared by chemical conjugation or by expression in an appropriatehost cell.

The terms “modulate,” “modulating,” and “modulation” refer to theincreasing or decreasing of a level of an active peptide. Modulation canoccur directly or indirectly.

The term “multimer,” as used herein, refers to a molecule comprisingmultiple peptide chains, that may be the same or different. The multimermay further comprise at least one optional linker to which at least twopeptides are covalently bound. A multimer may be, e.g., a dimer, trimer,or tetramer. A linker may be, e.g., a bis-thiol linker, a tris-thiollinker, a tetrathiol linker, a dicarboxylic acid linker, a tricarboxylicacid linker, a tetracarboxylic acid linker, an amine linker, a triaminelinker, or a tetraamine linker.

The term “peptide” refers to molecules comprising amino acids,including, e.g., L and D amino acids, linearly coupled through amidebonds. Peptides may additionally contain amino acid derivatives ornon-amino acid moieties such as, e.g., dipeptide mimetics. A peptide ofthe invention may range from, e.g., 2-100, 5-30, 7-50, 10-30, or 10-50amino acids (inclusive) in length, such as, e.g., from 11-35 aminoacids. Peptides may comprise further modifications, such as, e.g.,glycosylation, acetylation, phosphorylation, PEGylation, lipidation, orconjugation with an organic or inorganic molecule.

“Positively charged,” as used herein, refers to an amino acid, aminoacid mimetic, or chemical moiety that is positively charged at a pHgreater than 6, such as, e.g., from pH 6-8, 6-9, 7-8, or 7-9. Forexample, positively charged amino acids include lysine, arginine, and2,4-diaminobutyric acid.

The term “positively charged aromatic amino acid” refers to an aminoacid that is positively charged at a pH greater than 6, such as, e.g.,from pH 6-8, 6-9, 7-8, or 7-9. For example, positively charged aromaticamino acids include histidine, 4-aminophenylalanine, and4-guanylphenylalanine.

An amino acid sequence that is “substantially identical” to a givensequence may be, e.g., at least 60%, 64%, 70%, 75%, 76%, 80%, 82%, 85%,88%, 90%, 94%, 95%, 97%, 98%, or 99% identical to the given sequence. Itmay be derived from the given sequence by truncation, deletion,substitution, or addition of at least one amino acid, and/or may differfrom a given sequence by, e.g., the addition, deletion, or substitutionof at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

“Treat,” “treatment,” and “treating” refer to the reduction in severityor duration of a disease or condition; the amelioration of one or moresymptoms associated with a disease or condition; the provision ofbeneficial effects to a subject with a disease or condition, withoutnecessarily curing the disease or condition; or the prevention of adisease or condition.

A peptide “trimer” is a molecule comprising a first, second, and thirdpeptide chain, that may be the same or different. A peptide trimer mayfurther comprise at least one optional linker to which at least two ofthe peptides are covalently bound.

II. Peptides of the Invention

Peptides of the invention have been evaluated on the basis of relativeaffinity for FcRn and capacity to block the Fc portion of IgG frombinding to FcRn. Peptides that demonstrate the ability to bind FcRnand/or block the binding of FcRn to the Fc portion of IgG share certainsequence commonalities or features. Thus, one embodiment of theinvention provides a peptide capable of inhibiting the binding of the Fcportion of a human IgG to human Fc neonatal receptor, comprising thesequence:-Gly-X₆—X₇—X₈—X₉—X₁₀—X₁₁—

wherein:

-   -   X₆ is chosen from positively charged amino acids, aromatic amino        acids, positively charged aromatic amino acids, and analogs        thereof;    -   X₇ is chosen from phenylalanine and phenylalanine analogs,    -   X₈ and X₉ are each independently chosen from glycine, sarcosine,        aspartic acid, D-amino acids, α-aminoisobutyric acid, and        analogs thereof, or X₈, when taken together with X₉, forms a        dipeptide mimetic;    -   X₁₀ is chosen from amino acids and analogs thereof, or X₁₀, when        taken together with X₉, forms a dipeptide mimetic;    -   X₁₁ is chosen from tyrosine and tyrosine analogs; and        In certain embodiments, the peptide ranges from 7 to 50 amino        acids in length and binds to human FcRn, preventing FcRn from        binding to human IgG.

In one exemplary embodiment, a peptide of the invention comprises:Gly-X₆-Phe-X₈—X₉—X₁₀-Tyr.

In another embodiment, peptides of the invention can be represented bythe formula:R₁-Gly-X₆—X₇—X₉—X₁₀—X₁₁—R₂

wherein:

-   -   R₁ has the formula X₁—X₂—X₃—X₄        -   wherein            -   X₁ is chosen from hydrogen, acyl, and amino acid                protecting groups;            -   X₂ is absent or is chosen from an amino acid and                peptides of 2-15 amino acids in length;            -   X₃ is absent or is an amino acid that is capable of                forming a bridge with X₁₀, X₁₂ or X₁₃, wherein the                bridge is chosen from an amino terminus to carboxy                terminus bridge, a side chain to backbone bridge, and a                side chain to side chain bridge;            -   X₄ is absent or is chosen from an amino acid and                peptides of 2-15 amino acids in length;

—X₆, —X₇, —X₈, —X₉, —X₁₀, and —X₁₁ are as defined above; and

R₂ has the formula —X₁₂—X₁₃—X₁₄—X₁₅

wherein

-   -   X₁₂ is absent or is an amino acid;    -   X₁₃ is absent or is an amino acid;    -   X₁₄ is absent or is chosen from an amino acid and peptides of        2-15 amino acids in length; and    -   X₁₅ is an amino group or a carboxy protecting group.        The term “amino acid” as used to define the peptides of the        invention includes amino acid analogs.

In one embodiment, X₆ is a positively charged amino acid chosen fromlysine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid,arginine, guanylalanine, and analogs thereof. In another embodiment, X₆is an aromatic amino acid chosen from tyrosine, tryptophan,phenylalanine, and analogs thereof. In another embodiment, X₆ is apositively charged aromatic amino acid chosen from histidine,1-methylhistidine, 2-pyridylalanine, 3-pyridylalanine, 4-pyridylalanine,4-aminophenylalanine, 4-guanylphenylalanine, thiazolylalanine, andanalogs thereof. In another embodiment, X₆ is 4-guanylphenylalanine. Inanother embodiment, X₆ is chosen from histidine, 3-pyridylalanine,4-pyridylalanine, 4-guanylphenylalanine, and analogs thereof.

Exemplary histidine analogs are chosen from, but not limited to:diaminobutyric acid; thiazolylalanine; 2,3-diaminopropionic acid;guanylalanine; 2-pyridylalanine; 3-pyridylalanine; thienylalanine;ornithine; 4-guanylphenylalanine; 1-methylhistidine;4-aminophenylalanine; 2-pyrrolidinylalanine; 3-piperdylalanine; and4-piperidylalanine.

Exemplary phenylalanine derivatives may be chosen from, but not limitedto 4-aminophenylalanine; pentafluorophenylalanine; 2-pyridylalanine;3-pyridylalanine; 4-nitrophenylalanine; 1-napthylalanine;homophenylalanine; phenylglycine; 2-methylphenylalanine;3-methylphenylalanine; 4-methylphenylalanine; 2-chlorophenylalanine;3-chlorophenylalanine; 4-chlorophenylalanine; 3,3-di-phenylalanine;4,4-bi-phenylalanine; 4-t-butylphenylalanine; cyclohexylalanine;(4-aminoacetyl)-phenylalanine;L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;D-betamethylphenylalanine; and L-betamethylphenylalanine.

In one embodiment, X₁₀ is chosen from neutral and hydrophobic aminoacids, and analogs thereof.

In one embodiment, X₂ is chosen from an amino acid and peptides of 2 or3 amino acids in length. In one embodiment, X₂ includes at least onehydrophobic amino acid. In another embodiment, X₂ is an amino acid or apeptide of 2-15 amino acids in length, wherein the carboxy terminalamino acid is a hydrophobic amino acid.

In one embodiment, X₄ is an amino acid or a peptide of 2-15 amino acidsin length, wherein the carboxy terminal amino acid is N-methylated.

In one embodiment, the peptide is linear. In another embodiment, atleast one of X₁₀, X₁₂, or X₁₃ is an amino acid that is capable offorming a bridge with X₃, wherein the bridge is chosen from an aminoterminus to carboxy terminus bridge, a side chain to backbone bridge,and a side chain to side chain bridge. In one embodiment, the bridge isa side chain to side chain bridge, which can be a disulfide bridge, anether bridge, a thioether bridge, an alkene bridge, or an amide bridge.

In one embodiment, the side chain to side chain bridge is a disulfidebridge between: cysteine and cysteine; cysteine and homocysteine;cysteine and penicillamine; homocysteine and homocysteine; homocysteineand penicillamine; or penicillamine and penicillamine. In anotherembodiment, the side chain to side chain bridge is an amide bridgebetween: aspartic acid and lysine; aspartic acid and ornithine; asparticacid and 2,4-diaminobutyric acid; aspartic acid and 2,3-diaminopropionicacid; glutamic acid and lysine; glutamic acid and ornithine; glutamicacid and 2,4-diaminobutyric acid; or glutamic acid and2,3-diaminopropionic acid.

In one embodiment, the peptide comprises at least one cysteine (e.g., atX₃) or cysteine analog chosen from: homocysteine, D-cysteine, andpenicillamine.

In one embodiment, at least one of X₈ and X₉ is a D-amino acid or ischosen from: glycine, α-aminoisobutyric acid, and sarcosine.

In one embodiment, X₈, taken together with X₉, forms a dipeptide mimeticchosen from:

-   beta-alanine;-   4-aminobutanoic acid;-   5-aminopentanoic acid;-   3-(aminomethyl)benzoic acid;-   4-(aminomethyl)benzoic acid;-   3-(aminophenyl)acetic acid;-   4-(aminophenyl)acetic acid;

-   3-amino-2-oxo-1-piperidine-acetic acid;-   (3S)-3-amino-2-oxo-1-piperidine-acetic acid and    (3R)-3-amino-2-oxo-1-piperidine-acetic acid;-   (3S)-3-amino-2-oxo-1-azepine acetic acid and    (3R)-3-amino-2-oxo-1-azepine acetic acid;-   (3S)-3-amino-2-oxo-1-pyrrolidine acetic acid and    (3R)-3-amino-2-oxo-1-pyrrolidine acetic acid;-   (3R)-3-amino-1-carboxymethyl-valerolactam; and-   3-amino-N-1-carboxymethyl-2,3,4,5-tetrahydro-1H-[1]-benzazepine-2-one.

In some embodiments, the peptide comprises at least one phenylalaninephenylalanine analog chosen from: tryptophan; tyrosine;2-aminophenylalanine; 3-aminophenylalanine; 4-aminophenylalanine;pentafluorophenylalanine; 2-pyridylalanine; 3-pyridylalanine;4-nitrophenylalanine; 1-naphthylalanine; homophenylalanine;phenylglycine; 2-methylphenylalanine; 3-methylphenylalanine;4-methylphenylalanine; 2-chlorophenylalanine; 3-chlorophenylalanine;4-chlorophenylalanine; 3,3-diphenylalanine; 4,4′-biphenylalanine;4-t-butylphenylalanine; cyclohexylalanine; (4-aminoacetyl)phenylalanine;L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;D-beta-methylphenylalanine; and L-beta-methylphenylalanine.

In some embodiments, the peptide comprises at least one tyrosine analogchosen from: phenylalanine; 4-aminophenylalanine;4-methoxyphenylalanine; pentafluorophenylalanine; 2-pyridylalanine;3-pyridylalanine; 4-pyridylalanine; 4-nitrophenylalanine;2-nitrotyrosine; and 4-fluorophenylalanine.

In one embodiment, X₉ and X₁₀, taken together, form a dipeptide mimeticchosen from:

D,L-Friedinger's lactam

L,L-Friedinger's lactam

In certain embodiments, the peptide of the invention comprises at leastone histidine analog chosen from: 2,4-diaminobutyric acid;thiazolylalanine; 2,3-diaminopropionic acid; guanylalanine;2-pyridylalanine; 3-pyridylalanine; 4-pyridylalanine; thienylalanine;ornithine; lysine; arginine; 4-guanylphenylalanine; 1-methylhistidine;3-methylhistidine; 1,3-dimethylhistidine; 4-aminophenylalanine;2-pyrrolidinylalanine; 3-piperdylalanine; and 4-piperidylalanine.

In certain embodiments, the number of amino acids between those aminoacids forming a bridge ranges from 6-12. In other embodiments, eight ornine amino acids exist between amino acids forming a bridge.

In some embodiments the peptides of the invention are at least seven andas many as 50 amino acids long. In other embodiments the peptides arefrom 11 and 35 amino acids in length.

In certain embodiments, the peptides of the invention exist as amultimer, such as a dimer, a trimer or a tetramer. The peptides of amultimer may be the same or different.

In one embodiment, the peptide is a dimer, such as a dimer that is theproduct of reductive alkylation. In another embodiment, the dimer is theproduct of a reaction between individual peptide monomers and amultivalent linker. In one embodiment, the multivalent linker is chosenfrom thiol, acid, alcohol, and amine linkers. In another embodiment, thedimer is the product of an alkylation reaction, such as, e.g., thealkylation reaction of a thiol and an alkyl halide.

In one embodiment, the peptides are synthesized on the resin, and thenmultimerized (e.g., dimerized) by reaction with a multivalent linker,such as, e.g., an acid or amine multivalent linker, as described inExample 15.

In some embodiments, the peptide of the invention comprise at least 2 ofthe modifications described above.

In certain embodiments, the peptides of the invention comprise:

a) an amino acid sequence chosen from:

QRFCTGHFGGLYPCNGP, (SEQ ID NO:1) GGGCVTGHFGGIYCNYQ, (SEQ ID NO:2)KIICSPGHFGGMYCQGK, (SEQ ID NO:3) PSYCIEGHIDGIYCFNA, (SEQ ID NO:4) andNSFCRGRPGHFGGCYLF; (SEQ ID NO:5) and

b) an amino acid sequence that is substantially identically to one ormore of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ IDNO:5.

In some embodiments, the peptide comprises an amino acid sequence thatis at least 64%, at least 70%, at least 76%, at least 82%, at least 88%,at least 94%, or 100% identical to one or more of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In other embodiments,the peptide of the invention may comprise SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5 having at least one or as many asfive deletion, substitution, or addition mutations. All peptides of theinvention inhibit binding of human FcRn to IgG.

In some embodiments, a peptide of the invention may comprise SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 modified byat least one conservative amino acid substitution. In certainembodiments, a peptide of the invention may comprise SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 modified by at least oneamino acid substitution, wherein the amino acid is substituted with anaturally occurring amino acid. In some embodiments, a peptide of theinvention may have SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,or SEQ ID NO:5 modified by at least one amino acid substitution, whereinthe amino acid is substituted with a D-amino acid. In some embodiments,a peptide of the invention may have SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:5 modified by at least one amino acidsubstitution, wherein the amino acid is substituted with an N-methylatedamino acid. In some embodiments, a peptide of the invention may have SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 modifiedby at least one amino acid substitution, wherein the amino acid issubstituted with a non-naturally occurring amino acid. In certainembodiments, a peptide of the invention may have SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 modified by at least oneamino acid substitution, wherein the amino acid is substituted with anamino acid mimetic.

Alternatively, peptides of the invention may comprise an amino acidsequence chosen from Table 1 that is capable of binding to FcRn, therebyinhibiting FcRn binding to the Fc portion of an IgG molecule. Theinvention further includes peptides comprising a variant of thesequences listed in Table 1, wherein variants include, but are notlimited to: truncations; peptides that share at least 68%, 72%, 76%,80%, 84%, 88%, 92%, or 96%, identity with at least one of the peptidesof Table 1. Variants also include peptides comprising a sequence listedin Table 1 that contains at least one amino acid substitution, whereinthe amino acid is substituted with a naturally occurring amino acid, anon-naturally occurring amino acid, an amino acid analog, or an aminoacid mimetic.

TABLE 1 SEQ ID NO:6 AGQRFCTGHFGGLYPCNGPGTGGGK SEQ ID NO:7AGGGCVTGHFGGIYCNTQGTGGGK SEQ ID NO:8 AGKIICSPGHFGGMYCQGKGTGGGKSEQ ID NO:9 AGPSYCIEGHIDGIYCFNAGTGGGK SEQ ID NO:10AGNSFCRGRPGHFGGCYLFGTGGGK

In one embodiment, peptides of the invention may comprise an amino acidsequence listed in Table 1 that has 1, 2, 3, 4, 5, 6, or moreconservative amino acid substitutions. In another embodiment, peptidesof the invention may comprise an amino acid sequence listed in Table 1that has been substituted with 1, 2, 3, 4, 5, 6, or morenaturally-occurring amino acids.

In some embodiments, peptides of the invention may comprise an aminoacid sequence listed in Table 1 that has been substituted with 1, 2, 3,4, 5, 6, or more non-naturally-occurring amino acids. In anotherembodiment, peptides of the invention may comprise an amino acidsequence listed in Table 1 that has been substituted with 1, 2, 3, 4, 5,6, or more N-methylated amino acids. In another embodiment, peptides ofthe invention may comprise an amino acid sequence listed in Table 1 thathas been substituted with 1, 2, 3, 4, 5, 6, or more amino acids in theD-configuration. In yet another embodiment, peptides of the inventionmay comprise an amino acid sequence listed in Table 1 that has beensubstituted with 1, 2, 3, 4, 5, 6, or more amino acid mimetics.

Exemplary embodiments of the invention are provided below. Many of theseembodiments encompass the amino acid sequences listed in Table 1modified to include one or more amino acid substitutions. While theseembodiments provide examples of suitable substitutions, it should beunderstood that other substitutions that do not destroy the biologicalactivity of the peptides are encompassed by the invention.

In one exemplary embodiment, a peptide of the invention comprises theamino acid sequence of SEQ ID NO:6 modified with a Cysteine toPenicillamine substitution at position 6 and a Glycine to Sarcosinesubstitution at position 12 (wherein the positions of the amino acidsare based on the amino acid numbering in SEQ ID NO:6). In anotherembodiment, a peptide comprises the amino acid of SEQ ID NO:6 modifiedwith a Cysteine to Penicillamine substitution at position 6 and aLeucine to N-methylleucine substitution at position 13.

In another embodiment, a peptide of the invention may comprise the aminoacid sequence of SEQ ID NO:6 modified with a cysteine to Penicillaminesubstitution at position 6, substitutions of both the glycines atpositions 11 and 12, respectively for a single(3R)-amino-1-carboxymethyl-2-valerolactam; and a leucine toN-methylleucine substitution at position 13.

In one embodiment, a peptide of the invention may comprise the aminoacid sequence of SEQ ID NO:6 modified with a cysteine to penicillaminesubstitution at position 6, a glycine to sarcosine substitution atposition 12 and a leucine to N-methylleucine substitution at position13. In another embodiment, a peptide of the invention may comprise theamino acid sequence of SEQ ID NO:6 modified with a cysteine topenicillamine substitution at position 6, substitutions of both theglycines at positions 11 and 12, respectively for a single(3R)-amino-1-carboxymethylcaprolactam and a leucine to N-methylleucinesubstitution at position 13.

In one embodiment, a peptide of the invention may comprise the aminoacid sequence of SEQ ID NO:6 modified with a cysteine to penicillaminesubstitution at position 6, a histidine to 3-pyridylalanine substitutionat position 9, a glycine to sarcosine substitution at position 12 and aleucine to N-methylleucine at position 13. In another embodiment, apeptide of the invention may comprise the amino acid sequence of SEQ IDNO:6 modified with a cysteine to penicillamine substitution at position6, a histidine to 4-guanylphenylalanine substitution at position 9, aglycine to sarcosine substitution at position 12 and a leucine toN-methylleucine at position 13. In yet another embodiment, a peptide ofthe invention may comprise the amino acid sequence of SEQ ID NO:6modified with a cysteine to penacillamine substitution at position 6, ahistidine to 4-pyridylalanine substitution at position 9, a glycine tosarcosine substitution at position 12 and a leucine to N-methylleucineat position 13.

In certain embodiments, the peptides of the invention may exist as amultimer, such as a dimer, trimer, or tetramer. The peptides of themultimer may be the same or different. The peptides can be multimerizedas described above and in the ensuing Examples.

In one embodiment, the affinity of the peptides of the invention forFcRn will be represented by K_(D) with a value ranging from 50 fM to 1mM. In another embodiment, the affinity of the peptides of the inventionwill be represented by a K_(D) with a value ranging from 50 fM to 100μM, or a value ranging from 50 fM to 1 nM, or a value ranging from 1 pMto 1 nM. In another embodiment, a composition comprising at least one ofthe peptides of the invention will modulate the serum concentration ofIgG. In one embodiment, the peptides of the invention can block an IgGconstant region from binding to FcRn by binding to at least one aminoacid of FcRn that also specifically interacts with an IgG constantregion.

III. Methods of Making the Peptides of the Invention

1. General Methods of Synthesizing Peptides of the Invention

The peptides of the invention can be synthesized using techniques wellknown in the art. For example, peptides of the invention that arecomposed entirely of naturally occurring amino acids can be synthesizedrecombinantly in cells using polynucleotides encoding the peptide. See,e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory, N.Y. (1989) and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and WileyInterscience, N.Y. (1989). Alternatively, the peptides of the inventioncan be synthesized using known synthetic methods such as solid phasesynthesis. Synthetic techniques are well known in the art. See e.g.,Merrifield, Chemical Polypeptides, Katsoyannis and Panayotis eds. pp.335-61 (1973); Merrifield, J. Am. Chem. Soc. 85:2149 (1963); Davis etal., Biochem. Intl. 10:394 (1985); Finn et al., The Proteins (3d ed.)2:105 (1976); Erikson et al., The Proteins (3d ed.) 2:257 (1976).Standard Fmoc/tBu protocols may be used as described in W. C. Chan andP. D. White eds. Fmoc Solid Phase Peptide Synthesis: A PracticalApproach Oxford University Press Inc. New York (2000) and U.S. Pat. No.3,941,763. Alternatively, chimeric proteins of the invention can besynthesized using a combination of recombinant and synthetic methods. Incertain applications, it may be beneficial to use either a recombinantmethod, a synthetic method or a combination of recombinant and syntheticmethods.

2. Methods for Synthesizing Peptide Analogs of the Peptides of theInvention

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis,2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-di-substituted amino acids, N-alkyl amino acids, N-methyl amino acids,lactic acid, and other unconventional amino acids may also be suitablecomponents for polypeptides of the present invention. Examples ofunconventional amino acids include: aminoisobutyric acid,3-amino-1-carboxymethylvalerolactam, 4-guanyl-phenylalanine,5-aminopentanoic acid, 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, penicillamine, sarcosine, and other similar aminoacids and imino acids (e.g., 4-hydroxyproline). In the polypeptidenotation used herein, the left-hand direction is the amino terminaldirection and the right-hand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie;    -   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   3) acidic: Asp, Glu;    -   4) basic: H is, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol. 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in binding to a specific proteintarget.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included.

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In certain embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar peptides that are important foractivity or structure. In view of such a comparison, one can predict theimportance of amino acid residues in a peptide that correspond to aminoacid residues which are important for activity or structure in similarpeptides. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpeptides. One skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known to thoseskilled in the art. Such variants could be used to gather informationabout suitable variants. For example, if one discovered that a change toa particular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change may beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

According to certain embodiments, amino acid substitutions may be thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinities associatedwith the biological function, and/or (4) confer or modify otherphysicochemical or functional properties on such polypeptides. Accordingto certain embodiments, single or multiple amino acid substitutions (incertain embodiments, conservative amino acid substitutions) may be madein the naturally-occurring sequence (in certain embodiments, in theportion of the polypeptide outside the domain(s) forming intermolecularcontacts). In certain embodiments, a conservative amino acidsubstitution typically may not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples, Creighton, Ed., W.H. Freeman and Company, New York (1984);Introduction to Protein Structure, C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991); and Thornton et al., Nature354:105 (1991).

In certain embodiments, amino acid derivatives comprise covalentmodifications, including, but not limited to, chemical bonding withpolymers, lipids, or other organic or inorganic moieties. In certainembodiments, a chemically modified specific binding agent may havegreater circulating half-life than a specific binding agent that is notchemically modified. In certain embodiments, a chemically modifiedspecific binding agent may have improved targeting capacity for desiredcells, tissues, and/or organs. In certain embodiments, a derivativespecific binding agent is covalently modified to include one or morewater soluble polymer attachments, including, but not limited to,polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.See, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192; and 4,179,337. In certain embodiments, a derivative specificbinding agent comprises one or more polymers, including, but not limitedto, monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of such polymers.

3. Methods for Synthesizing Analogs of the Peptides of the Invention

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger, TINS p. 392 (1985); and Evans et al., J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylacetic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, and —CH₂ SO—, by methods well known in theart. Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used in certain embodiments to generate more stablepeptides. In addition, constrained peptides comprising a consensussequence or a substantially identical consensus sequence variation maybe generated by methods known in the art (Rizo and Gierasch, Ann. Rev.Biochem. 61:387 (1992), incorporated herein by reference), such as, forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

Peptide dimerization or oligomerization can enhance the avidity of apeptide sequence for a given receptor. See, for example, Johnson, et.al., Chem. Biol. 12:939 (1997). It is envisioned that dimers and higherorder multimers of the peptides of the invention could be synthesizedusing a variety of methods well known in the art. The dimers ormultimers could be synthesized directly on an automated peptidesynthesizer as a continuous peptide sequence. Alternatively, peptidemultimers could be synthesized by reacting individual peptide monomerswith a multivalent linker moiety. See, e.g., Rose, J. Am. Chem. Soc.116:30 (1994). As another example, peptide multimers may be synthesizedby incorporating branched linker groups prior to the synthesis of thepeptide sequence as in the synthesis of “multiple antigenic peptides”(MAP). D. Posnett et. al., J. Biol. Chem. 263:1719 (1988). The inventionprovides a novel method for forming these peptide dimers involvesreacting the N-termini of the peptides, while on resin, with a linkermolecule, such as, for example, succinic acid, so that adjacent peptideson the solid phase resin bead will react with each other and form adimer joined by the peptides N-termini. Subsequent cleavage from theresin provides an N-terminally linked peptide dimer.

4. Construction of Expression Vectors for the Expression of Peptides ofthe Invention

Nucleic acids encoding peptides of the invention may be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). Additional methods of nucleic acid synthesis areknown in the art. See, e.g., U.S. Pat. Nos. 6,015,881; 6,281,331;6,469,136. For recombinant production of peptides of the invention,polynucleotide sequences encoding the peptides are inserted intoappropriate expression vehicles, i.e. vectors which contains thenecessary elements for the transcription and translation of the insertedcoding sequence, or in the case of an RNA viral vector, the necessaryelements for replication and translation. The nucleic acids encoding thepeptides of the invention are inserted into the vectors in the properreading frame.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the DHFR gene or DHFR cDNA.Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80:2495 (1983).Selectable markers are reviewed by Thilly (Mammalian Cell Technology,Butterworth Publishers, Stoneham, Mass. (1986)) and the choice ofselectable markers is well within the level of ordinary skill in theart. Selectable markers may also be introduced into the cell on aseparate plasmid at the same time as the gene of interest, or they maybe introduced on the same plasmid. If on the same plasmid, theselectable marker and the gene of interest may be under the control ofdifferent promoters or the same promoter, the latter arrangementproducing a bicistronic message. Constructs of this type are known inthe art (for example, U.S. Pat. No. 4,713,339).

Expression elements in an expression systems vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in an expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter), and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g. heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g. the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells (e.g.metallothionein promoter) or from mammalian viruses (e.g. the adenoviruslate promoter; the vaccinia virus 7.5 K promoter) may be used; whengenerating cell lines that contain multiple copies of expressionproduct, SV40-, BPV- and EBV-based vectors may be used with anappropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the chimeric proteinsof the invention may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al., Nature 310:511-514 (1984)), or the coat proteinpromoter of TMV (Takamatsu et al., EMBO J. 6:307-311 (1987)) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al.,Science 224:838-843 (1984)) or heat shock promoters, e.g., soybeanhsp17,5-E or hsp17,3-B (Gurley et al., Mol. Cell. Biol. 6:559-565(1986)) may be used. These constructs can be introduced into plant cellsusing Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. Weissbach &Weissbach, Methods for Plant Molecular Biology, Academic Press, NY,Section VIII, pp. 421-463 (1988) and Grierson & Corey, Plant MolecularBiology, 2d Ed., Blackie, London, Ch. 7-9 (1988).

In one insect expression system that may be used to produce the chimericproteins of the invention, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example, the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e. virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see, e.g., Smith et al., J. Virol. 46:584 (1983);U.S. Pat. No. 4,215,051). Further examples of this expression system maybe found in Ausubel et al., eds., Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience (1989).

Another system which can be used to express the peptides of theinvention is the glutamine synthetase gene expression system, alsoreferred to as the “GS expression system” (Lonza Biologics PLC,Berkshire UK). This expression system is described in detail in U.S.Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g. region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. See e.g., Logan & Shenk, Proc. Natl. Acad.Sci. USA 81:3655 (1984). The vaccinia 7.5 K promoter may also be used.See e.g., Mackett et al., Proc. Natl. Acad. Sci. USA 79:7415 (1982);Mackett et al., J. Virol. 49:857 (1984); Panicali et al., Proc. Natl.Acad. Sci. USA 79:4927 (1982).

5. Expression of Peptides of the Invention in the Appropriate TargetCell

Expression vehicles may be transfected or co-transfected into a suitabletarget cell, to express the polypeptides of the invention. Transfectiontechniques known in the art include, but are not limited to, calciumphosphate precipitation (Wigler et al., Cell 14:725 (1978)),electroporation (Neumann et al., EMBO, J. 1:841 (1982)), and liposomebased reagents. A variety of host-expression vector systems may beutilized to express the chimeric proteins described herein includingboth prokaryotic and eukaryotic cells. These include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli) transformedwith recombinant bacteriophage DNA or plasmid DNA expression vectorscontaining an appropriate coding sequence; yeast or filamentous fungitransformed with recombinant yeast or fungi expression vectorscontaining an appropriate coding sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g. baculovirus) containingan appropriate coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g. cauliflower mosaic virus ortobacco mosaic virus) or transformed with recombinant plasmid expressionvectors (e.g. Ti plasmid) containing an appropriate coding sequence; oranimal cell systems, including mammalian cells (e.g., CHO, Cos, HeLacells).

6. Methods for Synthesizing Fusion Molecules that Comprise a Peptide ofthe Invention.

In some embodiments, peptides of the invention exist as a fusion proteincomprising the peptide of the invention and a fusion partner. In oneembodiment, the fusion partner confers properties, such as one or moreof extended half-life, stability, enhanced transport to the peptide ofthe invention, and/or can enhance efficacy in vivo or in vitro.

Additionally, heterologous polypeptides of the present invention can becombined with parts of the constant domain of immunoglobulins (IgG),resulting in chimeric polypeptides. These particular fusion moleculesfacilitate purification and show an increased half-life in vivo. Thishas been shown, for example, in chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. EP 0 394 827; Traunecker et al., Nature, 331:84-86(1988). Fusion molecules that have a disulfide-linked dimeric structuredue to the IgG part can in some instances be more efficient in bindingand neutralizing other molecules than, for example, a monomericpolypeptide or polypeptide fragment alone. See, for example,Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).

The invention also provides a polynucleotide encoding the fusionmolecule of any of the peptides described above. This polynucleotide maybe part of a vector that also comprises a regulatory sequence fortranscribing the polynucleotide. The invention further provides a hostcell comprising any of the fusion molecules described above, apolynucleotide encoding the fusion molecule of any of these peptides, ora vector that comprises a polynucleotide encoding the fusion molecule ofany of these peptides and a regulatory sequence for transcription of thepolynucleotide. The invention yet further provides a compositioncomprising any of the fusion molecules or nucleotides described above,and/or any of the vectors or host cells described above, and a buffer ora pharmaceutically acceptable carrier.

a. Fusions Comprising Antibody Fc Domains

In one embodiment, a peptide of the invention can be conjugated with theFc domain of IgG to increase its circulation half-life. In certainembodiments, the peptides are covalently linked to the Fc domain.Methods of making chimeric proteins comprising a Fc immunoglobulindomain both recombinantly and semi-synthetically are well-known to oneskilled in the art For example, an aldehyde may be incorporated into thepeptide derivative, and reacted with the Fc protein using a reductivealkylation reaction which is selective for the N-terminus of Fc.Kinstler, Adv. Drug Del. Rev. 54:477 (2002). Alternatively, a peptidethioester may be reacted with Fc bearing an N-terminal cysteine residueDawson and Kent, Ann. Rev. Biochem. 69:923 (2000).

Such peptide-Fc fusion derivatives may have an increased ability toblock the IgG-FcRn interaction due to the addition of two more bindingsites for FcRn through the Fc domain. Such peptide-Fc fusions may alsoprotect the peptide from degradation and thus enhance the in vivoefficacy of the peptide. Fusion proteins that have a disulfide-linkeddimeric structure due to the IgG portion can also be more efficient inbinding and neutralizing other molecules than the peptide of theinvention, for example, as described by Fountoulakis et al., J.Biochem., 270:3958-3964 (1995).

In embodiments where the peptide of the invention is part of a fusionprotein with a IgG Fc domain, the human Ig Fc may comprise a hinge, CH2,and CH3 domains of human IgG, such as human IgG1, IgG2, and IgG4. Theinvention also provides a fusion molecule comprising a peptide of theinvention and a variant Fc polypeptide or a fragment of a variant Fcpolypeptide, wherein the variant Fc comprises a hinge, CH2, and CH3domains of human IgG2 with a Pro331Ser mutation, as described in U.S.Pat. No. 6,900,292.

Suitable Fc domains for conjugating with peptides of the inventioninclude those having mutated amino acids at selected positions of the Fcregion to attenuate its effector functions (including antibody-dependentcell cytotoxicity and complement dependent cytotoxicity). For example,an Fc_(γ2) variant with the Pro331 Ser mutation has less complementactivity than natural Fc_(γ2) and does not bind to the Fc_(γ)R. IgG4 Fcis deficient in activating the complement cascade, and its bindingaffinity to Fc_(γ)R is about an order of magnitude lower than that ofthe most active isotype, IgG1. In one embodiment, a peptide of theinvention is conjugated to an Fc_(γ4) variant with Leu235Ala mutationwhich exhibits minimal effector functions as compared to the naturalFc_(γ4). In another embodiment, the peptide of the invention isconjugated to an Fc_(γ1) variant with Leu234Val, Leu235Ala and Pro331Ser mutations which also exhibit less effector function than naturalFc_(γ1).

b. Fusions Comprising Albumin

In certain embodiments, the peptides of the invention can be conjugatedto albumin-binding moieties. Such albumin-binding moiety-peptideconjugates may have longer in vivo half-lives and may thus require alower peptide doses to achieve the desired therapeutic effect. Chuang etal., Pharm. Res. 19:569 (2002); U.S. Pat. No. 6,685,179. Thus, oneembodiment of the invention provides a fusion molecule comprising thepeptide of the invention with an albumin fusion partner comprisingalbumin, one or more fragments of albumin, a peptide that binds albumin,and/or a molecule that conjugates with a lipid or other molecule thatbinds albumin.

Methods of making fusion proteins comprising albumin are known in theart. For example, peptides modified by hydrophobic aromatic cappingreagents have been shown to bind albumin non-covalently and extend thehalf-lives of peptides in rabbits. Zobel et. al., Bioorg. Med. Chem.Lett. 13:1513 (2003). As another example, peptides modified with thiolreactive groups have been shown to bind covalently to a single freecysteine residue on serum albumin. Kim et. al., Diabetes 52:751 (2003).

c. Fusions with Pegylated Moieties

In one embodiment, the peptide of the invention may be pegylated toinclude mono- or poly-(e.g., 2-4) PEG moieties. Pegylation may becarried out by any of the pegylation reactions known in the art. Methodsfor preparing a pegylated protein product will generally include (a)reacting a polypeptide with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions whereby thepeptide of the invention becomes attached to one or more PEG groups; and(b) obtaining the reaction product(s). In general, the optimal reactionconditions for the reactions will be determined case by case based onknown parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art and described in, for example, EP 0 401 384; Malik et al.,Exp. Hematol., 20:1028-1035 (1992); Francis, Focus on Growth Factors, 3(2):4-10 (1992); EP 0 154 316; EP 0 401 384; WO 92/16221; and WO95/34326. For example, the step of pegylating peptides of the inventionmay be carried out via an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule.

Thus, peptides of the invention include pegylated peptides wherein oneor more PEG groups are attached via acyl or alkyl groups. Such peptidesmay be mono-pegylated or poly-pegylated (for example, those containing2-6 or 2-5 PEG groups). The PEG groups are generally attached to thepeptide at the α- or ε-amino groups of amino acids, but it is alsocontemplated that the PEG groups could be attached to any amino group ofthe peptide that is sufficiently reactive to become attached to a PEGgroup under suitable reaction conditions.

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol (PEG) with a peptide of the invention.For acylation reactions, the polymer(s) selected typically have a singlereactive ester group. Any known or subsequently discovered reactive PEGmolecule may be used to carry out the pegylation reaction. An example ofa suitable activated PEG ester is PEG esterified to N-hydroxysuccinimide(NHS). As used herein, acylation is contemplated to include, withoutlimitation, the following types of linkages between a peptide of theinvention and a polymer such as PEG: amide, carbamate, urethane, and thelike. See, e.g., Chamow, Bioconjugate Chem., 5:133-140 (1994). Reactionconditions may be selected from any of those known in the pegylation artor those subsequently developed, but should avoid conditions such astemperature, solvent, and pH that would inactivate the peptide of theinvention.

Pegylation by acylation will generally result in a poly-pegylatedpeptide of the invention. The connecting linkage may be an amide. Theresulting product may be substantially only (e.g., >95%) mono, di- ortri-pegylated. However, some species with higher degrees of pegylationmay be formed in amounts depending on the specific reaction conditionsused. If desired, more purified pegylated species may be separated fromthe mixture (particularly unreacted species) by standard purificationtechniques, including among others, dialysis, salting-out,ultrafiltration, ion-exchange chromatography, gel filtrationchromatography and electrophoresis.

Pegylation by alkylation involves reacting a terminal aldehydederivative of PEG with a peptide of the invention in the presence of areducing agent. For the reductive alkylation reaction, the polymers)selected should have a single reactive aldehyde group. An exemplaryreactive PEG aldehyde is polyethylene glycol propionaldehyde, which iswater stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof. See,for example, U.S. Pat. No. 5,252,714.

7. Purification of Biologically Expressed Peptides of the Invention

Depending on the expression system used, the expressed peptide of theinvention is then isolated by procedures well-established in the art(e.g., affinity chromatography, size exclusion chromatography, ionexchange chromatography).

The expression vectors can encode for tags that permit for easypurification of the recombinantly produced chimeric protein. Examplesinclude, but are not limited to vector pUR278 (Ruther et al., EMBO J.2:1791 (1983)) in which DNA encoding a peptide of the invention isligated into the vector in frame with the lac z coding region so that ahybrid protein is produced; pGEX vectors may be used to express chimericproteins of the invention with a glutathione S-transferase (GST) tag.These proteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The vectors include cleavage sites(thrombin or Factor Xa protease or PreScission Protease™ (Pharmacia,Peapack, N.J.) for easy removal of the tag after purification.

To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the peptides of the inventionseparated by enzymatic cleavage sites. The resulting polypeptide can becleaved (e.g., by treatment with the appropriate enzyme) in order torecover the peptide units. This can increase the yield of peptidesdriven by a single promoter. When used in appropriate viral expressionsystems, the translation of each peptide of the invention encoded by themRNA is directed internally in the transcript, e.g., by an internalribosome entry site, IRES. Thus, the polycistronic construct directs thetranscription of a single, large polycistronic mRNA which, in turn,directs the translation of multiple individual polypeptides. Thisapproach eliminates the production and enzymatic processing ofpolyproteins and may significantly increase yield of the peptides of theinvention driven by a single promoter.

Host cells containing DNA constructs encoding the peptides of theinvention may be grown in an appropriate growth medium, i.e., a mediumcontaining nutrients required for the growth of cells. Nutrientsrequired for cell growth may include a carbon source, a nitrogen source,essential amino acids, vitamins, minerals and growth factors. Optionallythe media can contain bovine calf serum or fetal calf serum. In oneembodiment, the media contains substantially no IgG. The growth mediumwill generally select for cells containing the DNA construct by, forexample, drug selection or deficiency in an essential nutrient which iscomplemented by the selectable marker on the DNA construct orco-transfected with the DNA construct. Cultured mammalian cells aregenerally grown in commercially available serum-containing or serum-freemedia (e.g. MEM, DMEM). Selection of a medium appropriate for theparticular cell line used is within the level of ordinary skill in theart.

The peptides of the invention can be produced in a transgenic animal,such as a rodent, cow, pig, sheep, goat or other non-human animals thathave incorporated a foreign gene into their genome. Because this gene ispresent in germlne tissues, it is passed from parent to offspring.Exogenous genes are introduced into single-celled embryos (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438, 1985). Methods of producingtransgenic animals are known in the art, including transgenics thatproduce immunoglobulin molecules. Wagner et al., Proc. Natl. Acad. Sci.USA 78:6376 (1981); McKnight et al., Cell 34:335 (1983); Brinster etal., Nature 306:332 (1983); Ritchie et al., Nature 312:517 (1984);Baldassarre et al., Theriogenology 59:831 (2003); Robl et al.,Theriogenology 59:107 (2003); Malassagne et al., Xenotransplantation10(3):267 (2003).

8. Methods for Screening and Discovering Peptides that Bind FcRn andBlock the FcRn-IgG Interaction

Peptides binding to FcRn may be identified using phage displaylibraries. Phage display libraries may be readily generated as describedin Smith and Petrenko, Chem. Rev. 87:391 (1997). Alternatively, phagedisplay libraries may be acquired from a commercial source, such as,e.g., Dyax Corp. (Cambridge, Mass.). Depending on the screeningconditions, phage may be identified with a variety of differentproperties. To identify peptides that bind to FcRn (and thus competewith IgG for FcRn binding), a phage library may be screened for bindingto FcRn and by competition with IgG. Optionally, peptides that bind toalternate receptors may be eliminated from the library by incubating thephage library with one or more alternate receptors. Thus, phage thatbound the alternate receptor(s) would be depleted from the desired poolof phage. By sequencing the DNA of phage clones capable of binding toFcRn, peptides capable of binding to FcRn and inhibiting IgG-FcRnbinding may be identified.

Examples of other methods to identify FcRn-binding peptides include:mRNA display (Roberts and Szostak, Proc. Nat. Acad. Sci. USA 94:12297(1997), cell-based display (Boder and Wittrup, Nat. Biotechnol. 15:553(1997), and synthetic peptide libraries (Lam, Nature 354:82 (1991);Houghten et. al., Nature 354:84 (1991)).

9. Methods for Assaying Peptides that Bind to FcRn and Block theIgG:FcRn Interaction

A number of methods may be used to assess the ability of a peptide orpeptidomimetic to bind FcRn and block the FcRn:IgG interaction. Forexample, surface plasmon resonance (SPR) is a method well known in theart to evaluate binding events (Biacore AB, Uppsala, Sweden). Using thismethod, one of the binding partners (FcRn or IgG) is immobilized on theSPR sensor chip and while the other binding partner is passed over thechip, which is monitored for a resulting signal. In the same experiment,the peptide to be evaluated as a competitor of the interaction betweenIgG and FcRn is passed over the chip. Any decrease in signal may beinterpreted as a measure of the peptide's ability to block theinteraction between FcRn and IgG.

Other methods for assaying for possible peptide inhibitors of theIgG:FcRn interaction are also well known in the art. One such method isan IgG competition assay in a 96-well plate format. In this exampleassay, soluble human FcRn on a 96-well plate is exposed to IgG and atest peptide. Residual bound IgG, as detected by an anti-IgG antibodyand standard ELISA visualization reagents, provide a measure of thepeptide's ability to block the FcRn-IgG interaction.

The ability of a peptide to block IgG-FcRn binding may also be carriedout on cells transfected with DNA encoding a human FcRn to develop acell line capable of expressing human FcRn on its cell surface. Abinding competition assay may be applied where peptide inhibitors ofIgG-FcRn binding compete with a fluorescently labeled IgG molecule. Thelevel of residual IgG bound to the cells may be measured using, e.g., astandard fluorescent activated cell sorter (FACS).

d. Uses of the Peptides of the Invention

The peptides of the invention bind FcRn and inhibit the Fc portion ofthe IgG constant region from binding to FcRn resulting in increasedcatabolism of IgG in comparison to the catabolism of IgG in the absenceof peptides of the invention. In exemplary embodiments, the IgG constantregion is from the IgG1, IgG2, IgG3, or IgG4 subclasses. In particularembodiments, the IgG constant region is from IgG1, IgG2, or IgG4subclasses. The peptides of the invention are therefore useful to treatany disease or condition, where increased catabolism of IgG isdesirable. For example, peptides of the invention can be used to treatan autoimmune disease, an inflammatory disorder, cardiovascular diseasewith an inflammation-based etiology (e.g. arterial sclerosis),transplant rejection, and/or graft versus host disease (GVHD). Thepeptides of the invention can also be used to detect FcRn in a patientor a biological sample (e.g. a bodily fluid, a tissue or cell sample,cell culture supernatant). The peptides of the invention can also beused to purify FcRn from a biological sample (e.g. a bodily fluid, atissue or cell sample, cell culture supernatant).

Thus, the invention provides a method of regulating a disease statecharacterized by inappropriately expressed IgG antibodies or undesiredamounts or levels of IgG, comprising administering a therapeuticallyeffective amount of a peptide of the invention. In one embodiment, thedisease state is chosen from inflammatory diseases, autoimmune diseases,and cancer. In other embodiments, the invention provides methods forregulating a disease state by modulating the serum concentration of IgG.In certain embodiments, the methods of the invention may be employed totreat, prevent, or regulate an immune reaction to a therapeutic protein,such as, e.g., a erythropoietin, a lysosomal storage enzyme, or aclotting factor, such as, e.g., fibrinogen, prothrombin, factor V,factor VII, factor VIII, factor IX, factor X, factor XI, factor XII,factor XIII, or von Willebrand's factor. In other embodiments, themethods of the invention may be employed to treat, prevent, or regulatean immune reaction to a gene therapy vector.

1. Autoimmune Diseases

The peptides of the invention can be used to treat autoimmune diseasesincluding, but not limited to Alopecia Areata, Ankylosing Spondylitis,Antiphospholipid Syndrome, Autoimmune Addison's Disease, AutoimmuneHemolytic Anemia, Autoimmune Hepatitis, Autoimmune LymphoproliferativeSyndrome (ALPS), Autoimmune Thrombocytopenic Purpura (ATP), Behcet'sDisease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-DermatitisHerpetiformis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),Chronic Inflammatory Demyelinating Polyneuropathy, CicatricialPemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease,Degos' Disease, Dermatomyositis, Dermatomyositis-Juvenile, DiscoidLupus, Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis,Graves' Disease, Guillain-Barré Hashimoto's Thyroiditis, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgANephropathy, Insulin dependent Diabetes, Juvenile Arthritis, LichenPlanus, Lupus, Ménière's Disease, Mixed Connective Tissue Disease,Multiple Sclerosis, Myasthenia Gravis (MG), Pemphigus, PemphigusVulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis,Polyglandular Syndromes, Polymyalgia Rheumatica, Polymyositis andDermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis,Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever,Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome,Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant CellArteritis, Transplant Rejection, Ulcerative Colitis Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.

In one embodiment, the autoimmune disease is chosen from bullouspemphigoid, idiopathic thrombocytopenia purpura (ITP), myasthenia gravis(MG), pemphigus (e.g., pemphigus vulgaris), and transplant rejection.

In another embodiment, the peptides of the invention may be used incombination with steroids for immunosuppression.

2. Inflammatory Disorders

The peptides of the invention can be used to treat inflammatorydisorders including, but not limited to, asthma, ulcerative colitis andinflammatory bowel syndrome allergy, including allergicrhinitis/sinusitis, skin allergies (urticaria/hives, angioedema, atopicdermatitis), food allergies, drug allergies, insect allergies,mastocytosis, arthritis, including osteoarthritis, rheumatoid arthritis,and spondyloarthropathies.

3. Diseases or Conditions Requiring Administration of a TherapeuticProtein

Frequently, in diseases or conditions requiring administration of atherapeutic protein, the subject will develop antibodies against thetherapeutic protein, which, in turn, prevent the therapeutic proteinfrom be available for its intended therapeutic purpose. Accordingly, thepeptides of the invention can be used in combination with thetherapeutic protein to enhance the benefit of the therapeutic protein byreducing the levels of IgG; wherein, IgG antibodies are responsible forthe decreased bioavailability of a therapeutic protein.

Accordingly, one embodiment provides a method of regulating, treating,or preventing a condition, disease, or disorder resulting from an immuneresponse to a clotting factor comprising contacting a cell with atherapeutically effective amount of any of the peptides disclosedherein, wherein the clotting factor is chosen from fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor IX, factor X,factor XI, factor XII, factor XIII, or von Willebrand's factor. Thismethod may be used to regulate or treat, or prevent an immune responseto a clotting factor in a patient suffering, e.g., from hemophilia A orhemophilia B. In one embodiment, peptides of the present invention blockfactor VIII inhibitors. In another embodiment, the method may be used toregulate or treat, or prevent an immune response to, e.g., therapeuticerythropoietin in a patient suffering from pure red cell aplasia (PRCA).

4. Diseases or Conditions Requiring Gene Therapy

Obstacles to the successful implementation of gene therapy for thetreatment of a disease or condition also include the development ofantibodies specific to the therapeutic protein encoded by the transgeneas well as possibly to the vector used to deliver the transgene.Accordingly, the peptide of the invention can be administered incombination with gene therapy to enhance the benefit of the encodedtherapeutic protein by reducing the levels of IgG. These methods areparticularly useful in situations where IgG antibodies are responsiblefor the decreased bioavailability of a gene therapy vector or theencoded therapeutic protein. The gene therapy vector may be, e.g., aviral vector such as adenovirus and adeno associated virus. Diseasesthat can be treated using gene therapy include, but are not limited to,cystic fibrosis, hemophilia, PRCA, muscular dystrophy, or lysosomalstorage diseases, such as, e.g., Gaucher's disease and Fabry's disease.

5. In Vivo Imaging and Detection of FcRn

The peptides of the invention can also be used in assays to detect FcRn.In some embodiments, the assay is a binding assay that detects bindingof a peptide of the invention with FcRn. In these assays, either FcRn orthe peptides of the invention may be immobilized, while the other(either FcRn or the peptides of the invention) is passed over theimmobilized binding partner. Either FcRn or the peptides of theinvention may be detectably labeled. Suitable labels includeradioisotopes, including, but not limited to ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹¹¹In,¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁷Cs, ¹⁸⁶Re, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²⁴¹Am, ²⁴⁴Cm and 99 mTc-MDP; enzymes having detectable products (for example,luciferase, peroxidase, alkaline phosphatase, β-galactosidase, and thelike); fluorescers and fluorescent labels; for example, fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine; fluorescence emitting metals, forexample, ¹⁵²Eu, or others of the lanthanide series, attached to thepeptides of the invention through metal chelating groups such as EDTA;chemiluminescent compounds, for example, luminol, isoluminol, theromaticacridinium ester, acridinium salts, imidazole, and oxalate esteror; andbioluminescent compounds, for example, luciferin, or aequorin (greenfluorescent protein), specific binding molecules, for example, magneticparticles, microspheres, nanospheres, luminescent quantum dotnanocrystals, and the like.

Alternatively, specific-binding pairs may be used, involving, forexample, a second stage antibody or reagent that is detectably labeledand that can amplify the signal. For example, the peptides of theinvention can be conjugated to biotin, and horseradishperoxidase-conjugated streptavidin added as a second stage reagent.Digoxin and antidigoxin provide another suitable binding pair. In otherembodiments, a second stage antibody can be conjugated to an enzyme suchas peroxidase in combination with a substrate that undergoes a colorchange in the presence of the peroxidase. The absence or presence ofbinding between peptides of the invention and FcRn can be determined byvarious methods, including flow cytometry of dissociated cells,microscopy, radiography, fluorimetry, chromogenic detection, phosphorimaging, detection of chemiluminescence on film and scintillationcounting. Such reagents and their methods of use are well known in theart.

For in vivo diagnostic applications, specific tissues or even specificcellular disorders that may be characterized, at least in part, byexpression of FcRn, may be imaged by administration of a sufficientamount of a labeled peptide of the invention.

A wide variety of metal ions suitable for in vivo tissue imaging havebeen tested and utilized clinically. For imaging with radioisotopes, thefollowing characteristics are generally desirable: (a) low radiationdose to the patient; (b) high photon yield which permits a nuclearmedicine procedure to be performed in a short time period; (c) abilityto be produced in sufficient quantities; (d) acceptable cost; (e) simplepreparation for administration; and (f) no requirement that the patientbe sequestered subsequently. These characteristics generally translateinto the following: (a) the radiation exposure to the most criticalorgan is less than 5 rad; (b) a single image can be obtained withinseveral hours after infusion; (c) the radioisotope does not decay byemission of a particle; (d) the isotope can be readily detected; and (e)the half-life is less than four days (Lamb and Kramer, “CommercialProduction of Radioisotopes for Nuclear Medicine”, In Radiotracers ForMedical Applications, Vol. 1, Rayudu (Ed.), CRC Press, Inc., Boca Raton,pp. 17-62). In one embodiment, the metal is technetium-99m.

Accordingly, the invention provides a method of obtaining an image of aninternal region of a subject which comprises administering to a subjectan effective amount of a composition comprising at least one of thepeptides of the invention containing a metal in which the metal isradioactive, and recording the scintigraphic image obtained from thedecay of the radioactive metal. Likewise, the invention provides methodsfor enhancing an magnetic resonance (MR) image of an internal region ofa subject which comprises administering to a subject an effective amountof a composition comprising at least one of the peptides of theinvention containing a metal in which the metal is paramagnetic, andrecording the MR image of an internal region of the subject.

Other methods provided by this invention include a method of enhancing asonographic image of an internal region of a subject comprisingadministering to a subject an effective amount of a compositioncomprising at least one of the peptides of the invention containing ametal and recording the sonographic image of an internal region of thesubject. In this application, the metal may be any non-toxic heavy metalion. A method of enhancing an X-ray image of an internal region of asubject is also provided which comprises administering to a subject apeptide composition containing a metal, and recording the X-ray image ofan internal region of the subject. A radioactive, non-toxic heavy metalion may be used.

Peptides of the invention may be linked to chelators such as thosedescribed in U.S. Pat. No. 5,326,856. The peptide-chelator complex maythen be radiolabeled to provide an imaging agent for diagnosis ortreatment of diseases or conditions involving the regulation of IgGlevels. The peptides of the invention may also be used in the methodsthat are disclosed in U.S. Pat. No. 5,449,761 for creating aradiolabeled peptide for use in imaging or radiotherapy.

6. Dosing and Treatment Modalities

The peptides of the invention can be administered intravenously,subcutaneously, intra-muscularly, orally, sublingually, buccally,sublingually, nasally, rectally, vaginally or via pulmonary route. Thepeptides of the invention can be implanted within or linked to abiopolymer solid support that allows for the slow release of thechimeric protein to the desired site.

The dose of the peptide of the invention will vary depending on thedisease or condition to be treated, the severity of the disease orconditions, the subject, including their gender, age, weight and desiredoutcome and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg body weight. In one embodiment, thedosing range is 1-10,000 μg/kg. In another embodiment, the dosing rangeis 10-1,000 μg/kg. In another embodiment, the dosing range is 100-500μg/kg. The peptide of the invention can be administered continuously orat specific timed intervals. In vitro assays may be employed todetermine optimal dose ranges and/or schedules for administration. Othereffective dosages can be readily determined by one of ordinary skill inthe art through routine trials establishing dose response curves, forexample, the amount of the peptides of the invention necessary toincrease or decrease the level of IgG can be calculated from in vivoexperimentation. Those of skill will readily appreciate that dose levelscan vary as a function of the specific compound, the severity of thesymptoms, and the susceptibility of the subject to side effects, andpreferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means. For example, in order tocalculate the dose of peptides of the invention, those skilled in theart can use readily available information with respect to the amountnecessary to have the desired effect, depending upon the particularagent used.

7. Pharmaceutical Compositions

The invention also relates to a pharmaceutical composition comprising atleast one of the peptides of the invention and a pharmaceuticallyacceptable carrier or excipient. Examples of suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences by E. W.Martin. Examples of excipients can include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid, for example as a syrup ora suspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle.

For buccal and sublingual administration the composition may take theform of tablets or lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from a pressurized pack or nebulizer (e.g. in PBS), with asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous or intramuscular) by bolus injection.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multidose containers with an added preservative. Thecompositions can take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., pyrogen free water.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Peptides of the invention may be linked to chelators such as thosedescribed in U.S. Pat. No. 5,326,856. The peptide-chelator complex maythen be radiolabeled to provide an imaging agent for diagnosis ortreatment of diseases or conditions involving the regulation of IgGlevels. The peptides of the invention may also be used in the methodsthat are disclosed in U.S. Pat. No. 5,449,761 for creating aradiolabeled peptide for use in radiotherapy.

8. Purification of FcRn

The peptides of the invention can also be used to purify FcRn. In someembodiments, the peptide is covalently attached to an appropriatechromatographic matrix to form an efficient FcRn separation media. Asolution containing FcRn is then passed over the chromatographic matrixresulting in the non-covalent binding of FcRn to the immobilized bindingpartner. Solutions containing FcRn may be from biological samples suchas a bodily fluid, a tissue or cell sample, cell culture supernatant.The FcRn is purified by washing the immobilized peptide:FcRn complexwith a suitable solution to remove impurities and then releasing theFcRn from the chromatographic matrix with a suitable elution solution.

Peptides of the invention can be attached to suitable chromatographicmatrices using a number of chemical approaches well known to thoseskilled in the art. For example, peptides of the invention can beattached to matrices containing suitably reactive groups, such asthiols, amines, carboxylic acids, alcohols, aldehydes, alkyl halides,N-alkyl-maleimides, N-hydroxy-succinimidyl esters, epoxides,hydroxylamines, hydrazides.

In other embodiments, the peptides of the invention can be modified tocontain chemical moieties or peptide sequences that bind non-covalentlyto an appropriate chromatographic matrix. For example, the peptidescould be modified with a biotin moiety and could be non-covalently boundto a chromatographic matrix containing an avidin protein. Alternatively,the modified peptide could be incubated with the FcRn solution and theresulting mixture passed over the appropriate chromatographic matrix toisolate the FcRn:peptide complex.

Examples of similar uses of peptides for affinity purification can befound in Kelley et al., “Development and Validation of an AffinityChromatography Step Using a Peptide Ligand for cGMP Production of FactorVIII”, In Biotechnology and Bioengineering, Vol. 87, No. 3, WileyInterScienc, 2004, pp. 400-412 and in U.S. Pat. No. 6,197,526.

EXAMPLES

The Examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The Examples are not intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (forexample, amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric pressure.

Example 1 Expression of Soluble Human FcRn (shFcRn)

Soluble human FcRn cDNA was cloned, expressed and purified as describedin the literature using the glutamine synthetase expression system inChinese hamster ovary (CHO) cells See U.S. Pat. No. 5,623,053. A stopcodon was placed after amino acid position 274 in the protein sequenceof human FcRn in order to remove the transmembrane region.

Example 2 Transfection of HEK 293 Cells with Human FcRn

Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, Va.) weretransfected using the SuperFect Transfection Reagent (Qiagen, Valencia,Calif.) according to the manufacturer's recommended protocol. The fulllength FcRn cDNA construct depicted in FIG. 1 (C. M. Story et al., J.Exp. Med. 180:2377-2381 (1994), N. E. Simister et al., Eur. J. Immunol.26:1527-1531 (1996)) was originally cloned into pcDNA6 (Invitrogen,Carlsbad, Calif.) as the plasmid vector in order to generate the FcRnexpression vector, FcRn:pcDNA6. The Human β₂m cDNA construct, alsodepicted in FIG. 1 was originally cloned into pcDNA3 (Invitrogen) as theplasmid vector to generate the human β₂m expression vector, β₂m:pcDNA3(D. Gussow et. al., J. Immunol. 139:3132-3138 (1987)).

The day before transfection, HEK293 cells were seeded at 0.5-2.5×10⁶cells per 100 mm dish and incubated at 37° C. and 5% CO₂ for 16 hours incDMEM. The composition of cDMEM contains: 1 L DMEM (Invitrogen#11995-065); 10 ml of 1 M HEPES, pH 7.55; 10 ml MEM amino acid solution(Invitrogen #11130-051); 10 ml MEM non-essential amino acid solution(Invitrogen #11140-050); 10 ml of 100 mM sodium pyruvate (Invitrogen#11360-070); 10 ml of Penicillin Streptomycin liquid (Invitrogen#15140-148); 10 ml L-glutamine solution (Invitrogen #25030-081); 1 ml2-mercaptoethanol solution in 55 mM Dulbecco phosphate buffered saline(DPBS) (Invitrogen #21985-023); 100 ml heat-inactivated fetal bovineserum (FBS) (Invitrogen). On the day of the transfection, 5 μg of theFcRn:pcDNA6 construct and 5 μg of β₂m:pcDNA3 DNA were added to 290 μL ofDMEM (Invitrogen). The solution was mixed for a few seconds andcentrifuged. Then, 60 μL of SuperFect Transfection Reagent (Qiagen) wasadded to the DNA solution and vortexed for 10 seconds. The DNA/SuperFectsolution was incubated for 5 to 10 minutes at room temperature, duringwhich time the media from the cell-containing dish was aspirated and thecells washed once with 4 ml of PBS. After the 5 to 10 minute incubationof the DNA/SuperFect, 3 ml of complete growth medium (cDMEM) was addedto the DNA/SuperFect solution, the solution was mixed, and immediatelyadded to the cells in the 100 mm dish.

The cells were incubated with the DNA/SuperFect solution for 2 to 3hours at 37° C. and 5% CO₂. The media containing the DNA/SuperFectsolution was removed from the cells the cells were washed 3 times withPBS and fresh cDMEM was added to the cells. After a 48 hour incubation,the medium was assessed by immunoblot analysis to determine if transientexpression of the FcRn/β₂m complex had occurred. In addition, the cellswere passaged at a ratio of 1:4 into cDMEM containing 250 μg/L Geneticin(Invitrogen) as an antibiotic and 5 μg/L Blasticidin to select forBlasticidin resistant stable transfectants. After 4 weeks of antibioticselection, surviving cells were seeded into 96-well tissue cultureplates at a density of 1 cell per well. Ultimately 12 clones wereselected and each expanded and checked for expression by immunoblotanalysis for FcRn and β₂m. This analysis identified the FcRn andβ₂m-expressing 293 clone 11 as possessing the highest level ofexpression and was thus used in subsequent assays.

Example 3 Screening of Phage Libraries for FcRn-IgG Inhibitors

Peptides capable of inhibiting the binding of the IgG Fc portion to FcRnwere identified by screening filamentous phage display librarieslicensed from Dyax Corp. (Cambridge, Mass.). More specifically, thefollowing three libraries were used in combination; TN-9-IV, TN10-X,TN-11-I and TN-12-I were used in the screen. The total number ofindividual viable phage contained in each library was reflected by thenumber of transformants established for each library when the librarieswere expressed in E. coli and plated at a clonal dilution as describedby the Dyax protocol. The number of transformants for TN-9-IV, TN10-X,TN-11-I and TN-12-I was 3.2×10⁹, 2×10⁹, 2.7×10⁹ and 1.4×10⁹,respectively. Another way to refer to the absolute number of viablephages in a given volume is by stating the plaque forming units (pfu)per unit volume.

A. Buffers Used in Phage Screening

The following buffers were used for the screening of FcRn-bindingpeptides.

-   -   1. NZCYM Broth: 10 g NZ Amine-A; 5 g sodium chloride; 5 g Bacto        Yeast Extract (Difco); 1 g Casamino acids; 1 g magnesium sulfate        anhydrous powder: ingredients were dissolved in 800 ml of water,        adjusted to pH 7.5 with 1 N sodium hydroxide and then brought up        to a total volume of 1 L with water and autoclaved for 20 min.    -   2. Binding buffer (BB): PBS, pH 6 plus 10 mM EDTA.C. NZCYM-T:        NZCYM broth plus 12.5 μg/ml Tetracycline.    -   3. HBSS-E: Hank's Balanced Saline Solution (Invitrogen) plus 10        mM EDTA (Invitrogen).    -   4. Min A Salts: 10.5 g K₂HPO₄ (potassium phosphate dibasic); 4.5        g KH₂PO₄ (potassium phosphate monobasic); 1.0 g (NH₄)₂SO₄        (ammonium sulfate) and 0.5 g sodium citrate dissolved in 1 L        water.    -   5. LB Broth: 10 g Bacto Tryptone; 5 g Bacto yeast extract; 10 g        sodium chloride dissolved in 1 L water and autoclaved for 20        min.    -   6. CBS pH 2: 50 mM sodium citrate; 150 mM sodium chloride:        buffer was brought to pH 2 with HCl and filter sterilized.    -   7. LB Agar: 30 g Bacto Tryptone; 15 g Bacto yeast extract; 30 g        sodium chloride dissolved in 3 L water and autoclaved for 20        minutes.    -   8. LB Soft Agar: 20 g Bacto Tryptone; 10 g Bacto yeast extract;        20 g sodium chloride; 14 g Bacto agar dissolved in 2 L water        using mild heat without boiling.    -   9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 7

B. Screening Protocol: Round 1

Approximately 100 random library equivalents of each library were pooledaccording to their titer, which meant; 24 μL of TN9-IV (1.3×10¹⁰⁻pfu/μL), 12.5 μL of TN10-X (1.6×10¹⁰ pfu/μL), 225 μL of TN11-I (1.2×10⁹pfu/μL), and 48.7 μL of TN12-I (2.9×10⁹ pfu/μL) were mixed with 189 μLPBS, 75 μL of ice-cold 17% polyethylene glycol (PEG) (average molecularweight: 8000 Da, Sigma-Aldrich, St. Louis, Mo.) and 75 μL of 3 M sodiumchloride and incubated on ice for 30 minutes. One T75 flask of 293 clone11 cells (Example 2) was split at a ratio of 1:3 with HBSS-E. The cellswere transferred to a 1 ml microcentrifuge tube, washed once with coldbinding buffer and the supernatant removed. The cells were incubatedwith the phage for 1.5 hours at 4° C. on a rotator. After theincubation, the cells were washed five times with 1 ml of ice-cold BBfollowed each time by centrifugation at 1400 rpm for 2 minutes. Thestrongly bound phage were eluted by adding 66 μM human IgG (Calbiochem,San Diego, Calif.) that had been dialyzed into BB. The phage-IgG mix wasincubated with the cells for 1 hour at 4° C. Following a centrifugationstep (1400 rpm spin for 2 min.), the cell pellet washed first with 200μL of 66 μM IgG, centrifuged (1400 rpm spin for 2 min.) and washed afinal time with 100 μl IgG. The IgG washes were combined with the IgGelution for final volume of 500 μl. The phage in the eluent were titeredand amplified as described below.

C. Phage Titer

Phage solutions were diluted in 100-fold steps. Typically 2 μl of phagesolution was added to 198 μL of NZCYM broth in a serial manner toachieve dilutions of up to 10⁻¹⁰. Diluted phage were added to a cultureof XL1 Blue MRF′ E. coli cells when the XL1 Blue MRF′ E. coli cells werebeing grown in log phase and reached an optical density of 0.5 at A600(UV absorbance at 600 nm). The culture was incubated at room temperaturefor 10 minutes. Afterwards, 0.5 ml of the infected cells were added to3.5 ml of molten top agar (a 50/50 mix of LB broth and LB agar) atapproximately 55° C. and spread onto a standard agar plate and incubatedovernight at 37 degrees. The titer was calculated from a platecontaining 30 to 300 plaques. For a plate containing 50 plaques, platedfrom a 10⁻⁸ phage dilution, the calculations would be performed asfollows: 50 plaques/500 μL infected cells×10-fold dilution duringinfection×10⁸ phage dilution=10⁸ plaque-forming units per μL.

When necessary for subsequent phage ELISA and sequencing analysis,individual agar plugs containing phage plaques were picked withautoclaved Pasteur pipets. Plugs were deposited in 96-well sterileround-bottom tissue culture plates (Greiner), to which 100 μL per wellTE were added. Phage were eluted from the plaques for 2 hours at 37° C.or overnight at 4° C.

D. Phage Amplification

A culture of XL1 blue MRF′ E. coli cells were grown in NZCYM broth-T,from a 1/100 dilution of a saturated overnight culture until the culturereached an optical density of 0.5 at A600. The cells were concentratedby centrifuging them for 15 minutes at 3500 rpm, followed byresuspension in Min A salts to 1/20 of the original volume. The phageeluted from cells after a round of selection were diluted to a 1 mlfinal volume in Min A salts and added to 1 ml of the concentratedbacterial culture. After a 15 minute incubation in a 37° C. water bath,the phage-cell mix was added to 2 ml 2×NZCYM broth and spread on a largeNUNC plate with NZCYM plus 50 μg/ml Ampicillin until dry. Plates wereincubated for 14 to 18 hours at 37° C. Colonies that formed overnightwere scraped gently with a spreading bar in the presence of 20 ml ofPBS. PBS-containing bacteria and phage were collected in a centrifugetube. Bacteria remaining on the plate were scraped again in the presenceof 10 ml PBS and collected. A final 10 ml PBS rinse was applied to theplate, and pooled together with all scraped material. The bacterialcells were pelleted by centrifugation (15 minutes at 3500 rpm), and theclear supernatant was decanted into another centrifuge tube, clarifiedagain, and finally decanted again. Then, a 0.15 mL volume of 17% PEG+3MNaCl was added to the supernatant, which was mixed and stored overnightat 4° C. The precipitated phage collected by centrifugation (8500×g for30 minutes), after which, the supernatant was discarded. The phagepellet was resuspended in a small volume of PBS, clarified with a briefspin, and precipitated again with a 0.15 volume of 17% PEG+3M NaCl. Thefinal phage pellet was resuspended in PBS and titered in preparation forthe next round of selection.

E. Round 2

The amplified phage library was diluted such that only 10 random libraryequivalents were diluted into 1 ml of binding buffer. One third of a T75flask of untransfected 293 cells washed once with cold binding buffer. Asubtraction step included to remove phage from the library thatexpressed peptides capable of binding to cells that did not express FcRnwas performed twice by incubating the phage with the untransfected cellsfor 15 minutes. The supernatant was recovered. Then, one third of a T75flask of 293 clone 11 cells washed once with cold binding buffer andincubated with the phage for 1.5 hours at 4° C. in a rotator. The cellswere washed and centrifuged (1400 rpm spin for 2 min.) five times with 1ml cold binding buffer and the strongly bound phage were eluted with 200μL of 66 μM human IgG (dialyzed in binding buffer) by incubating thephage-cell-IgG mixture for 1 hour at 4° C. After centrifugation (1400rpm spin for 2 min.), the supernatant was collected and the pelletwashed with 200 μL of 66 uM IgG, followed by a 100 μL wash of 66 uM IgG.The phage in the eluent were titered and amplified as described below inthe sections labeled phage titer and phage amplification.

F. Round 3

This round was performed as described above for Round 2. At thecompletion of Round 3, the phage in the eluent were titered and assayedfor IgG-FcRn inhibitors using the phage ELISA.

G. Phage ELISA

The following steps were carried out to identify, by enzyme linkedimmunosorbent assay (ELISA), phages encoding peptides that were able tobind FcRn. First, the following solutions were prepared:

Buffer A: PBS+0.1% Tween+0.5% BSA.

Buffer B: 100 mM MES, pH 5.5+150 mM NaCl+0.1% Tween.

Buffer C: 50 mM MES, pH 6.0+150 mM NaCl+0.1% Tween

An XL1 blue MRF′ E. coli culture for the propagation of a phage thatdemonstrated the ability to bind FcRn was grown to an optical density of0.5 at A600 from a 1:100 dilution of an overnight culture. Then, 10 μlof each phage plaque eluate that was prepared as described above wereadded to 30 μl of the XL1 blue MRF′ E. coli cells into wells of a96-well plate and incubated for 15 minutes at room temperature. Then,130 μl of NZCYM broth containing 50 μg/ml of Ampicillin were added toeach well and the plates were incubated overnight at 37° C.

A Streptavidin-coated, BSA-blocked microtiter plate (Pierce) wasprepared by rinsing it with 200 μl per well of buffer A, and coating itovernight at 4° C. with 1 mg/ml of biotinylated soluble human FcRn(Example 4, section A), in buffer A. The FcRn-containing buffer wasdiscarded and the plate was rinsed twice with buffer C. Then, 70 μl ofbuffer B was added to each well of the plate, followed by the additionof 30 μl of a bacterial culture containing phage. After 1 hour at roomtemperature, the plate washed five times with 200 μl of buffer C. Then,100 μl of buffer C containing a 1:10000 dilution of an HRP-conjugatedanti-M13 antibody (Amersham Pharmacia) was added to each well. The platewas incubated at room temperature for one hour. Then, the plates werewashed 9 times with buffer C, developed with 1 step TMB (KPL), stoppedafter 5-15 minutes with 2M sulfuric acid and read at 450 nm with aSpectra Max Plus plate reader (Molecular Devices).

H. PCR Amplification of Phage DNA

Phage eluted from plaques in TE were amplified for sequencing by usingthe PCR Core System II kit per the manufacturer's instructions(Promega). Then, 5 ml of eluted phage was added to a reaction mixcontaining 200 μM each dNTP, 500 nM of primer 3PCRUP(5′-CGGCGCAACTATCGGTATCAAGCTG-3′) (SEQ ID NO:11), 500 nM of primer3PCRDN (5′-CATGTACCGTAACACTGAGTTTCGTC-3′) (SEQ ID NO:12), 1× Taq DNAPolymerase Buffer (10×: 500 mM KCl, 100 mM Tris-HCl pH 9.0 at 25° C., 1%Triton X-100, 15 mM MgCl₂), and 1.25 units Taq DNA Polymerase enzyme.The reactions were subjected to the following program on a MJ ResearchPCT-200 thermal cycler: 5 minutes at 94° C.; 30 cycles consisting of 15seconds at 94° C., 30 seconds at 55° C., and 60 seconds at 72° C.,followed by 7 minutes at 72° C. The resulting product was purified usingthe QiaQuick PCR Prep kit (Qiagen) according to manufacturer'sinstructions, quantified by absorbance at A260, and sequenced usingprimer 3SEQ-80 (5′-GATAAACCGATACAATTAAAGGCTCC-3′) (SEQ ID NO:13).

Sequencing of phage that was amplified following the 3 rounds ofscreening revealed the DNA sequences that encoded the amino acidsequences provided in FIG. 1. These “phage hits” were used collectivelyto identify a consensus peptide sequence, defined by the amino acidsequence: G-H-F-G-G-X-Y (SEQ ID NO: 14).

Example 4 Peptide-IgG Competition ELISA

In order to determine whether the peptides of the invention that werederived from the screening of the filamentous phage display librarieswere also able to block the binding of IgG to FcRn, the following ELISAassay was devised and performed.

A. Biotinylation of shFcRn

A solution of soluble human FcRn (shFcRn) in Tris buffer was dialyzedtwice, each time for 3 hours in 2 liters of PBS, pH 8.0. The quantity ofrecovered shFcRn was determined by measuring the absorbance at 280 nm.The concentration of shFcRn was obtained by multiplying the absorbancereading by the extinction coefficient for shFcRn, which is: ε=85880 M⁻¹cm⁻¹. Biotinylation of shFcRn was accomplished by treating the dialyzedshFcRn with a 2-fold-molar excess of Sulfo-NHS-LC-Biotin (Invitrogen,Carlsbad, Calif.) for 2 hours at 4° C. Afterwards, theshFcRn-Sulfo-NHS-LC-Biotin reaction mixture was dialyzed twice in 2 L ofcold PBS, followed by another absorbance reading to determine theconcentration of the remaining protein. The biotinylated shFcRn wasstored at 4° C. with 0.1% sodium azide until needed.

B. Peptide-IgG Competition ELISA Assay

96-well ReactiBind Neutravidin-coated plates blocked with BSA (Pierce,Rockford, Ill.) were washed twice with 200 μl/well of Buffer A (BufferA: PBS pH 7.4 (Gibco, 14040), 0.5% BSA IgG-free, 0.05% Tween-20). Thewells were coated with 100 μl/well of 1 μg/ml biotinylated-shFcRn inBuffer A. The plate was sealed and incubated at 37° C. for 2 hours.Afterwards, the plate washed with 200 μl/well of Buffer B (Buffer B: 100mM MES pH 6, 150 mM NaCl, 0.5% BSA IgG-free (Jackson ImmunoResearch,West Grove, Pa.), 0.05% Tween-20). Then, 50 μl/well of 6 nM human IgG(Calbiochem, San Diego, Calif.) in Buffer B as well as 50 μl/well of thevarious peptide competitors (at various concentration) were added, sothat the final concentration of IgG in the well was 3 nM. To allow formixing, the plate was rocked for 2 minutes, sealed and incubated at 37°C. for 2 hours. Following the incubation, the liquid was aspirated fromthe plate and 100 μl/well of a 1:10 000 dilution ofPeroxidase-conjugated goat anti-human IgG F(ab′) fragment-specificF(ab′)₂ fragment (Jackson ImmunoResearch, West Grove, Pa.) in Buffer Bwas added. The plate was covered, incubated for 30 minutes at roomtemperature and washed 4 times with 200 μl/well of ice-cold buffer B.SureBlue TMB substrate solution (100 μl/well, KPL, Gaithersburg, Md.)was added and the plate was allowed to incubate at room temperatureuntil color developed, which took 5 to 10 minutes. Once color developed,100 μl/well of TMB stop solution (KPL, Gaithersburg, Md.) was added andthe absorbance was measured at 450 nm. The data was plotted asabsorbance vs. peptide concentration to derive the inhibitoryconcentration 50% (IC₅₀) values.

Example 5 Peptide-IgG Competition FACS Assay

In addition to using the ELISA approach described in Example 4 todetermine whether the peptides of the invention that were derived fromthe screening of the filamentous phage display libraries were also ableto block the binding of IgG to FcRn on cells, the following fluorescenceactivated cell sorting (FACS) assay was devised and performed.

A. Labeling of Synagis® with Alexa-Fluor-488

Humanized IgG1 (Synagis®, MedImmune, Gaithersburg, Md.) was labeled withthe Alexa Fluor 488 Protein Labeling Kit (Molecular Probes/Invitrogen,Carlsbad, Calif.) according to the manufacturer's suggested protocol.Briefly, 50 μl of 1 M sodium bicarbonate, pH 9.0 was added to 500 μl ofa 2 mg/ml solution of IgG in PBS. This protein solution was added to theAlexa Fluor 488 succinimidyl ester (dry powder) and incubated at roomtemperature for 1 hour. The protein was purified by size-exclusionchromatography using the kit component column (Bio-Rad BioGel P-30 Finesize exclusion purification resin). The sample was loaded onto thecolumn and eluted with PBS. The first colored band contained the labeledprotein. The degree of labeling was determined by measuring theabsorbance of the eluted IgG at 280 nm and 494 nm. The protein molarconcentration was determined using the formula: protein concentration(M)=[A₂₈₀−(A₄₉₄×0.11)×dilution factor]/203,000. In addition, the formulaused to derive the moles of dye per mole of protein was: =A₄₉₄× dilutionfactor/71,000× protein concentration (M). Typically, 4-7 moles ofAlexa-Fluor 488 were incorporated per mole of IgG.

B. IgG-Peptide Competition FACS Assay Using 293 Clone 11 Cells

In preparation for the assay, HEK 293 clone 11 cells (Example 2) incomplete DMEM media (Gibco, Carlsbad, Calif.) containing 5 μg/mlBlasticidin and 250 μg/ml G418 (Gibco, Carlsbad, Calif.) were spun downand resuspended in Buffer C (Buffer C: Dulbecco's PBS (Gibco, Carlsbad,Calif.) containing 10 mM EDTA (Gibco)) at a concentration of 3×10⁶cells/ml. Cells (0.1 ml) were pipetted into each well of a 96-well assayplate and the plates were centrifuged at 2600 RPM for 5 min using aSorvall RT7 benchtop centrifuge. The supernatants were gently decantedand the plate was blotted on a paper towel. Peptide competitors (90 μl)solubilized in Buffer C at various concentrations were added to theplate and mixed with a multi-channel pipette. 10 μl of Alexa 488-labeledSynagis® was added to each well on the plate, such that the finalconcentration of Alexa 488-labeled Synagis® was 100 nM. The plate waswrapped in foil, placed on ice for one hour and subsequently centrifugedat 2600 rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge followedby a single wash with 100 μl of Buffer C and a second centrifugationstep. The cells were resuspended in 200 μl of Buffer C and analyzed on aBeckman Coulter EPICS XL flow cytometer.

Example 6 Methods for the Determination of Equilibrium Binding Constants(K_(D)) for Peptides Using Surface Plasmon Resonance (SPR)

The following steps were performed to cross-link soluble human orcynomolgus FcRn to the dextran surface of a CM5 sensor chip (Biacore AB,Uppsala, Sweden) by an amine coupling reaction involving1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)(Biacore AB, Uppsala, Sweden) and N-hydroxysuccinimide (NHS) (BiacoreAB, Uppsala, Sweden) as recommended by Biacore (BIAapplicationsHandbook, version AB, section 4.2, Biacore AB, Uppsala, Sweden). TheFcRn protein was diluted in 50 mM sodium acetate, pH 4.5 (Biacore AB,Uppsala, Sweden) to a concentration of 10 to 30 μg/ml and used to coatone flow cell on the sensor chip. Residual sites on the FcRn flow cellwere blocked with 1 M ethanolamine hydrochloride pH 8.5 (Biacore AB,Uppsala, Sweden). A control flow cell was blocked with ethanolamine forreference subtraction. For analysis of the monomeric peptides, FcRn wascoated to a final density of 4000-5000 response units (RU). For analysisof the peptide dimers, FcRn was coated to a density of 2000-2500 RU. AllSPR measurements were performed using a BIACORE 3000 Instrument (BiacoreAB Uppsala, Sweden). For measurements done at either pH 6 or pH 7.4,experiments were performed in 50 mM phosphate, 100 mM sodium chloride,0.01% surfactant P20 (Biacore AB, Uppsala, Sweden).

A. Representative Procedure for the Determination of Binding Constant ofMonomeric Peptides

Ten, 2-fold dilutions of the peptide were injected over the FcRn-CM5chip at a rate of 20 μl/min for 2 min. The peptide was dissociated fromthe chip for 2.5 minutes with buffer. Any remaining peptide was removedfrom the chip with a 30 second injection of HBS-P buffer (Biacore AB,Uppsala, Sweden) at a rate of 30 μl/min. Sensorgrams were generated andanalyzed using BiaEval software version 3.1 (Biacore AB, Uppsala,Sweden). The equilibrium RU observed for each injection was plottedagainst concentration. The equilibrium K_(D) values were derived byanalysis of the plots using the steady state affinity model included inthe BiaEval software.

B. Representative Procedure for Determination of Binding Constant ofDimeric Peptides

Ten, 2-fold dilutions of the peptide were injected over the FcRn-CM5chip at a rate of 30 μl/min for 10 min. Peptides were dissociated fromthe chip for 10 minutes with buffer. Any remaining peptide was removedfrom the chip with two, 60 second injections of a solution containing 50mM Tris-hydrochloride, 100 mM NaCl, 0.01% surfactant P20 pH 9.0 at 100μl/min.

Sensorgrams were generated and analyzed using BiaEval software version3.1 (Biacore AB, Uppsala, Sweden). The equilibrium RU observed for eachinjection was plotted against concentration. The equilibrium K_(D)values were derived by analysis of the plots using the steady stateaffinity model included in the BiaEval software.

Example 7 Synthesis of Monomeric Peptides Containing Disulfide Bonds

Synthesis of monomeric peptides was performed using solid-phase peptidesynthesis either manually with a fritted round bottom flask or by usingan Advanced Chemtech 396-omega synthesizer (Advanced Chemtech,Louisville, Ky.). Standard Fmoc/tBu protocols were used (W. C. Chan andP. D. White eds., Fmoc Solid Phase Peptide Synthesis: A PracticalApproach Oxford University Press Inc. New York (2000)), in combinationwith a Rink amide resin (Novabiochem, San Diego, Calif.) or PAL-PEG-PS(Applied Biosystems, Foster City, Calif.) to yield C-terminal amidesupon cleavage. The coupling reagents were2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and N-hydroxybenzotriazole (HOBt) (Novabiochem, San Diego,Calif.). The base was diisopropylethylamine (DIEA) (Sigma-Aldrich, St.Louis, Mo.), and N,N-dimethylformamide (DMF) was the solvent (EMScience, Kansas City, Mo.). The typical synthesis cycle involved 2×10minute deprotection steps with 20% piperidine in DMF, 2×30 minute aminoacid couplings with HOBt/HBTU and a 10 minute capping step with aceticanhydride/HOBt. Peptides were cleaved from the resin by treatment for 2hours with 95% trifluoroacetic acid; 2.5% thanedithiol 1.5%triisopropylsilane and 1% water and precipitated with ice-cold ether,centrifuged and triturated three times with ether.

Crude cysteine-containing peptides were oxidized to their correspondingdisulfides by dissolving the peptides to a concentration of 1 mg/ml in a4:1 mixture of acetic acid and water (EM Science, Kansas City, Mo.). Tenmolar equivalents of iodine (1M solution in water, Sigma-Aldrich, St.Louis, Mo.) were added to the solution and the reaction mixture wasmixed for one hour at room temperature. The reaction was stopped by theprogressive addition of 1 M sodium thiosulfate (Sigma-Aldrich, St.Louis, Mo.) until a clear solution was obtained. The reaction mixturewas concentrated in vacuo and subsequently purified using a WatersPrep600 reversed phase HPLC system (Millford, Mass.) equipped with a 250mm×21.2 mm Phenomenex (Torrence Calif.) C18 column. The eluent chosenfor the HPLC purification step was a gradient of acetonitrile in watercontaining 0.1% (w/v) TFA. Appropriate fractions were collected, pooledand lyophilized. Peptide identity and purity was confirmed by reversedphase analytical HPLC in combination with a 250 mm×2 mm column(Phenomenex, Torrence, Calif.) coupled with electrospray massspectrometry (Mariner ES-MS) (Applied Biosystems, Foster City, Calif.).

Table 2 provides a listing of the original phage peptide sequencesderived from the screen of the peptide expression library used toidentify peptides with a high affinity for human FcRn and the ability toblock the IgG-FcRn interaction. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6.

TABLE 2 Original Phage Peptide Sequences K_(D) K_(D) IC₅₀ (pH 6) pH 7.4Sequence μM μM μM SEQ ID NO:6 AGQRFCTGHFGGLYPCNGPGTGGGK 36 5.7 45SEQ ID NO:7 AGGGCVTGHFGGIYCNTQGTGGGK 33 5.2 34.7 SEQ ID NO:8AGKIICSPGHFGGMYCQGKGTGGGK 64 22 78 SEQ ID NO:9 AGPSYCIEGHIDGIYCFNAGTGGGK49 8.8 76 SEQ ID NO:10 AGNSFCRGRPGHFGGCYLFGTGGGK 33 9.4 93

Table 3 provides a listing of truncations of the SEQ ID NO:6 peptide.The effect of the truncations on the binding parameters of thesepeptides with human FcRn are shown. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6.

TABLE 3 Truncations of SEQ ID NO:6 K_(D) K_(D) IC₅₀ (pH 6) pH 7.4Sequence μM μM μM SEQ ID NO:6 AGQRFCTGHFGGLYPCNGPGTGGGK 36 5.7  45SEQ ID NO:1 QRFCTGHFGGLYPCNGP 26 5.1  30 SEQ ID NO:17 CTGHFGGLYPCNGP 23934 nd SEQ ID NO:18 QRFCTGHFGGLYPC 27 4.2  26 SEQ ID NO:19 CTGHFGGLYPC110 20 320 SEQ ID NO:20 TGHFGGLYP >250 >250 nd SEQ ID NO:21RFCTGHFGGLYPCNGP 24 2.9  78 SEQ ID NO:22 FCTGHFGGLYPCNGP 67 11 120SEQ ID NO:23 QRFCTGHFGGLYPCNG 34 4.6  69 SEQ ID NO:24 QRFCTGHFGGLYPCN 316.1  73

Table 4 provides a listing of SEQ ID NO:1-derived peptides and peptideanalogs, in which single amino acids have been substituted with alanine(an alanine scan). The effect of the substitutions on the bindingparameters of these peptides with human FcRn are shown. Column 1contains the peptide identifier. Column 2 contains the amino acidsequence of the peptides. Column 3 contains the IC₅₀ of each peptide asdetermined by the IgG competition ELISA outlined in Example 4. Columns 4and 5 contain the K_(D) of each peptide as determined at pH 6 and pH7.4, respectively, by the Biacore analysis outlined in Example 6.

TABLE 4 Alanine Scan of SEQ ID NO:1 K_(D) K_(D) IC₅₀ (pH 6) pH 7.4Sequence μM μM μM SEQ ID NO:1 QRFCTGHFGGLYPCNGP 26 5.1  30 SEQ ID NO:25QAFCTGHFGGLYPCNGP 23 7.7 nd SEQ ID NO:26 QRACTGHFGGLYPCNGP 95 28 ndSEQ ID NO:27 QRFCAGHFGGLYPCNGP 30 4.9 nd SEQ ID NO:28QRFCTAHFGGLYPCNGP >125 >250 nd SEQ ID NO:29 QRFCTGAFGGLYPCNGP >125 >250nd SEQ ID NO:30 QRFCTGHAGGLYPCNGP >125 >250 nd SEQ ID NO:31QRFCTGHFAGLYPCNGP >125 230 200 SEQ ID NO:32 QRFCTGHFGALYPCNGP >125 120110 SEQ ID NO:33 QRFCTGHFGGAYPCNGP 107 26  81 SEQ ID NO:34QRFCTGHFGGLAPCNGP >125 >250 nd SEQ ID NO:35 QRFCTGHFGGLYACNGP 96 14 100SEQ ID NO:36 QRFCTGHFGGLYPCAGP 30 8 nd

Table 5 provides a listing of SEQ ID NO:1-derived peptides and peptideanalogs in which substitutions of cysteines with cysteine derivativeshave been performed. The effect of the substitutions on the bindingparameters of these peptides with human FcRn are shown. Column 1contains the peptide identifier. Column 2 contains the amino acidsequence of the peptides. Column 3 contains the IC₅₀ of each peptide asdetermined by the IgG competition ELISA outlined in Example 4. Columns 4and 5 contain the K_(D) of each peptide as determined at pH 6 and pH7.4, respectively, by the Biacore analysis outlined in Example 6.

TABLE 5 Cysteine Derivatives of SEQ ID NO:1 K_(D) K_(D) Sequence IC₅₀(pH 6) pH 7.4 (SEQ ID NOS 1 & 37-47) μM μM μM SEQ ID NO:1QRF-C-TGHFGGLYP-C-NGP 26 5.1 30 Peptide No. 27 QRFCTGHFGGLYP-hC-NGP 213.9 Peptide No. 28 QRF-hC-TGHFGGLYP-hC-NGP 20 3.8 Peptide No. 29QRF-c-TGHFGGLYP-C-NGP >125 150 Peptide No. 30 QRF-C-TGHFGGLYP-c-NGP 12531 Peptide No. 31 QRF-c-THGFGGLYP-c-NGP >500 200 Peptide No. 32QRF-Pen-TGHFGGLYP-C-NGP 2 0.25 Peptide No. 33 QRF-C-TGHFGGLYP-Pen-NGP 182.7 Peptide No. 34 QRF-Pen-TGHFGGLYP-Pen-NGP 2 0.37 Peptide No. 69QRF-Pen-TGHFGGLYP-hC-NGP 2 0.31 Peptide No. 70 QRF-hC-TGHFGGLYP-Pen-NGP16 2.1 Peptide No. 295 QRF-Pen-TGHFG-p-LYP-Pen-NGP 1.6 0.28 * “Pen” =L-penicillamine; “hC” = L-homocysteine;

Table 6 provides a listing of SEQ ID NO:1 and Peptide No. 32 derivedpeptides in which single amino acids have been substituted for N-methylamino acids. The effect of the substitutions on the binding parametersof these peptides with human FcRn are shown. Column 1 contains thepeptide identifier. Column 2 contains the amino acid sequence of thepeptides. Column 3 contains the IC₅₀ of each peptide as determined bythe IgG competition ELISA outlined in Example 4. Columns 4 and 5 containthe K_(D) of each peptide as determined at pH 6 and pH 7.4,respectively, by the Biacore analysis outlined in Example 6.

TABLE 6 N-Methyl Scan of SEQ ID NO:1 and Peptide No. 32 K_(D) K_(D)Sequence IC₅₀ (pH 6) pH 7.4 (SEQ ID NOS 1 & 48-60) μM μM μM SEQ ID NO:1QRFCTGHFGGLYPCNGP 26 5.1 30 Peptide No. 196 QRFC-NMeAla-GHFGGLYPCNGP 16918 Peptide No. 32 QRF-Pen-TGHFGGLYP-C-NGP 2 0.25 Peptide No. 108QRF-Pen-T-Sar-HFGGLYP-C-NGP >125 88 Peptide No. 192RF-Pen-TG-NMeHis-FGGLYPC >250 nd Peptide No. 110QRF-Pen-TGH-NMePhe-GGLYPCNGP >125 >250 Peptide No. 111QRF-Pen-TGHF-Sar-GLYPCNGP 27 2 Peptide No. 112 QRF-Pen-TGHFG-Sar-LYPCNGP0.9 0.11 Peptide No. 113 QRF-Pen-TGHFGG-NMeLeu-YPCNGP 1.6 0.086Peptide No. 114 QRF-Pen-TGHFGGL-NMeTyr-PCNGP >125 92 Peptide No. 146RF-Pen-TGHFGG-NMeLeu-YPCNGP 2.1 0.059 0.28 Peptide No. 147RF-Pen-TGHFG-Sar-YPCNGP 1.0 0.058 0.35 Peptide No. 187QRF-Pen-TGHFG-Sar-NMeLeu-YPCNGP 0.42 0.046 0.23 Peptide No. 235RF-Pen-TGHFG-Sar-NMeLeu-YPC 0.49 0.031 0.17 * Sar = sarcosine; NMeAla =N-methyl alanine; “NMe” prefix denotes N-methyl amino acid

Table 7 provides a listing of truncations of Peptide No. 32-derivedpeptide derivatives. The effect of the truncations on the bindingparameters of these peptides with human FcRn are shown. Column 1contains the peptide identifier. Column 2 contains the amino acidsequence of the peptides. Column 3 contains the IC₅₀ of each peptide asdetermined by the IgG competition ELISA outlined in Example 4. Columns 4and 5 contain the K_(D) of each peptide as determined at pH 6 and pH7.4, respectively, by the Biacore analysis outlined in Example 6.

TABLE 7 Truncations of Peptide No. 32 K_(D) K_(D) Sequence IC₅₀ (pH 6)pH 7.4 (SEQ ID NOS 61-69) μM μM μM Peptide No. 32 QRF-Pen-TGHFGGLYPCNGP2 0.25 1.2 Peptide No. 82 F-Pen-TGHFGGLYPC 1.7 0.31 5 Peptide No. 83NH₂-F-Pen-TGHFGGLYPC 3.1 0.29 12 Peptide No. 99 RF-Pen-TGHFGGLYPC 2.00.17 3.4 Peptide No. 141 QRF-Pen-TGHFGpLYPC 1.5 0.19 Peptide No. 142RF-Pen-TGHFGpLYPC 1.5 0.14 Peptide No. 143 F-Pen-TGHFGpLYPC 1.7Peptide No. 144 RF-Pen-TGHFGpLYPCNGP 1.5 Peptide No. 145F-Pen-TGHFGpLYPCNGP 3.1 * “Pen” = L-penicillamine

Table 8 provides a listing of Peptide No. 32-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: Gly-Gly-Leu. Column 1 contains the peptide identifier. Column2 contains the amino acid sequence of the peptides. Column 3 containsthe IC₅₀ of each peptide as determined by the IgG competition ELISAoutlined in Example 4. Columns 4 and 5 contain the K_(D) of each peptideas determined at pH 6 and pH 7.4, respectively, by the Biacore analysisoutlined in Example 6.

TABLE 8 Analogs of Peptide No. 32 at Gly-Gly-Leu K_(D) K_(D) SequenceIC₅₀ (pH 6) pH 7.4 (SEQ ID NOS 70-98) μM μM μM Peptide No. 32QRF-Pen-TGHF-GG-LYP-C-NGP 2 0.25 1.2 Peptide No. 40QRF-Pen-TGHF-G-p-LYPCNGP 1.4 0.23 1.1 Peptide No. 41QRF-Pen-TGHF-G-r-LYPCNGP 8.1 0.83 8.8 Peptide No. 42QRF-Pen-TGHF-G-h-LYPCNGP 12 2 20 Peptide No. 43 QRF-Pen-TGHF-G-i-LYPCNGP18 2.2 41 Peptide No. 44 QRF-Pen-TGHF-G-f-LYPCNGP 13 1.7 100Peptide No. 45 QRF-Pen-TGHF-G-y-LYPCNGP 13 1.5 31 Peptide No. 46QRF-Pen-TGHF-G-Aib-LYPCNGP 2.4 0.48 5.3 Peptide No. 47QRF-Pen-TGHF-d-G-LYPCNGP 3.1 0.58 4.9 Peptide No. 48QRF-Pen-TGHF-p-G-LYPCNGP 5 0.79 21 Peptide No. 49QRF-Pen-TGHF-r-G-LYPCNGP 4.1 0.31 Peptide No. 50QRF-Pen-TGHF-h-G-LYPCNGP 3.6 0.41 Peptide No. 51QRF-Pen-TGHF-i-G-LYPCNGP 9.4 2.6 Peptide No. 52 QRF-Pen-TGHF-f-G-LYPCNGP2.8 0.51 Peptide No. 53 QRF-Pen-TGHF-y-G-LYPCNGP 3.2 0.32 Peptide No. 54QRF-Pen-TGHF-Aib-G-LYPCNGP 17 5.2 Peptide No. 74QRF-Pen-TGHF-G-a-LYPCNGP 2 0.48 12 Peptide No. 75QRF-Pen-TGHF-a-G-LYPCNGP 4.5 0.49 4.5 Peptide No. 148QRF-Pen-TGHF-a-a-LYPCNGP 4.5 0.45 Peptide No. 149QRF-Pen-TGHF-a-p-LYPCNGP 3.7 0.43 Peptide No. 150QRF-Pen-TGHF-f-p-LYPCNGP 5.9 0.72 Peptide No. 151QRF-Pen-TGHF-f-a-LYPCNGP 4.3 0.41 Peptide No. 152QRF-Pen-TGHF-p-p-LYPCNGP 21 3.3 Peptide No. 153QRF-Pen-TGHF-f-G-NMeLeu-YPCNGP 1.3 0.24 Peptide No. 154QRF-Pen-TGHF-a-G-NMeLeu-YPCNGP 3.2 0.23 Peptide No. 155QRF-Pen-TGHF-f-G-P-YPCNGP 39 18.3 Peptide No. 202QRF-Pen-TGHF-p-P-LYPCNGP >250 >100 Peptide No. 203QRF-Pen-TGHF-f-P-LYPCNGP 22 3.8 Peptide No. 189QRF-Pen-TGHF-a-Sar-LYPCNGP 1.7 0.19 * “Sar” = sarcosine; “Aib” =aminoisobutyric acid

Table 9 provides a listing of Peptide No. 32-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: Arg-Phe-Penicillamine. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6.

TABLE 9 Analogs of Peptide No. 32 at Arg-Phe-Pen K_(D) K_(D) SequenceIC₅₀ (pH 6) pH 7.4 (SEQ ID NOS 99-102) μM μM μM Peptide No. 32QR-F-Pen-TGHFGGLYPCNGP 2 0.25 1.2 Peptide No. 96 QR-f-Pen-TGHFGGLYPCNGP11.4 1.8 Peptide No. 97 QR-Y-Pen-TGHFGGLYPCNGP 2.4 0.31 Peptide No. 98QR-W-Pen-TGHFGGLYPCNGP 1.5 0.29

Table 10 provides a listing of Peptide No. 32-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: Penicillamine-Thr-Gly. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6.

TABLE 10 Analogs of Peptide No. 32 at Pen-Thr-Gly K_(D) K_(D) SequenceIC₅₀ (pH 6) pH 7.4 (SEQ ID NOS 99-102) μM μM μM Peptide No. 32QRF-Pen-T-GHFGGLYPCNGP 2 0.25 1.2 Peptide No. 296 QRF-Pen-H-GHFGGLYPCNGP3 0.15 0.96 Peptide No. 195 QRF-Pen-G-GHFGGLYPCNGP 7.7 0.76Peptide No. 213 QRF-Pen-(NMeAla)-GHFGGLYPCNGP 5.5 1.0 * “NMeAla” =N-methyl alanine

Table 11 provides a listing of Peptide No. 187-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: Phe-Gly-Sarcosine. Column 1 contains the peptide identifier.Column 2 contains the amino acid sequence of the peptides. Column 3contains the IC₅₀ of each peptide as determined by the IgG competitionELISA outlined in Example 4. Columns 4 and 5 contain the K_(D) of eachpeptide as determined at pH 6 and pH 7.4, respectively, by the Biacoreanalysis outlined in Example 6.

TABLE 11 Analogs of Peptide No. 187 at Phe-Gly-Sar K_(D) K_(D) SequenceIC₅₀ (pH 6) pH 7.4 (SEQ ID NOS 107-119) μM μM μM Peptide No. 187QRF-Pen-TGHF-G-Sar-NMeLeu-YPCNGP 0.42 0.046 0.23 Peptide No. 188QRF-Pen-TGHF-a-Sar-NMeLeu-YPCNGP 6.5 0.73 Peptide No. 235RF-Pen-TGHF-G-Sar-NMeLeu-YPC 0.49 0.031 0.17 Peptide No. 217RF-Pen-TGHF-f-Sar-NMeLeu-YPC 11 1.4 Peptide No. 218RF-Pen-TGHF-v-Sar-NMeLeu-YPC >50 13 Peptide No. 219RF-Pen-TGHF-l-Sar-NMeLeu-YPC 4 0.47 Peptide No. 220RF-Pen-TGHF-w-Sar-NMeLeu-YPC 11 2.7 Peptide No. 240RF-Pen-TGHF-t-Sar-NMeLeu-YPC 71 4.8 Peptide No. 241RF-Pen-TGHF-s-Sar-NMeLeu-YPC 23 1.1 Peptide No. 242RF-Pen-TGHF-d-Sar-NMeLeu-YPC 33 2.6 Peptide No. 243RF-Pen-TGHF-n-Sar-NMeLeu-YPC 29 2.1 Peptide No. 244RF-Pen-TGHF-e-Sar-NMeLeu-YPC 6.4 0.58 Peptide No. 245RF-Pen-TGHF-g-Sar-NMeLeu-YPC 4.5 0.36 * “Sar” = sarcosine; “Aib” =aminoisobutyric acid

Table 12 provides a listing of Peptide No. 32-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: His-Phe-Gly. Column 1 contains the peptide identifier. Column2 contains the amino acid sequence of the peptides. Column 3 shows thechemical structure of Phe analog side-chain. Column 4 contains the IC₅₀of each peptide as determined by the IgG competition ELISA outlined inExample 4. Column 5 contains the K_(D) of each peptide as determined atpH 6 by the Biacore analysis outlined in Example 6.

TABLE 12 Analogs of Peptide No. 32 at His-Phe-Gly K_(D) Sequence IC₅₀(pH 6) (SEQ ID NOS 120-145) Phe Analog Side-Chain μM μM Peptide No. 32QRF-Pen-TGH-F-GGLYPCNGP

2 0.25 Peptide No. 55 QRF-Pen-TGH-(4-amino-Phe)-GGLYPCNGP

13 1 Peptide No. 56 QRF-Pen-TGH-(4-methoxy-Phe)-GGLYPCNGP

100 18 Peptide No. 57 QRF-Pen-TGH-(pentafluoro-Phe)-GGLYPCNGP

120 70 Peptide No. 58 QRF-Pen-TGH-(2-pyridylalanine)-GGLYPCNGP

90 1.2 Peptide No. 59 QRF-Pen-TGH-(3-PyridylAla)-GGLYPCNGP

60 19 Peptide No. 60 QRF-Pen-TGH-(4-nitro-Phe)-GGLYPCNGP

>125 84 Peptide No. 61 QRF-Pen-TGH-(1-napthylalanine)-GGLYPCNGP

13 2.2 Peptide No. 62 QRF-Pen-TGH-(2-napthylalanine)-GGLYPCNGP

90 11 Peptide No. 88 QRF-Pen-TGH-(2-MePhe)-GGLYPCNGP

1 0.20 Peptide No. 89 QRF-Pen-TGH-(3-MePhe)-GGLYPCNGP

4.1 0.67 Peptide No. 90 QRF-Pen-TGH-(4-MePhe)-GGLYPCNGP

1.7 0.20 Peptide No. 92 QRF-Pen-TGH-(homoPhe)-GGLYPCNGP

80 7.8 Peptide No. 93 QRF-Pen-TGH-(Cha)-GGLYPCNGP

31 4.5 Peptide No. 94 QRF-Pen-TGH-(PheNHAc)-GGLYPCNGP

>125 270 Peptide No. 95 QRF-Pen-TGH-W-GGLYPCNGP

26 2.7 Peptide No. 102 QRF-Pen-TGH-(phenylGly)-GGLYPCNGP

>125 >250 Peptide No. 103 QRF-Pen-TGH-(Tic)-GGLYPCNGP

>125 >250 Peptide No. 104 QRF-Asp-TGH-(2MePhe)-GGLYP-Lys-NGP¹

11 Peptide No. 221 RF-Pen-TGH-(2-Cl-Phe)-GGLYPC

4 Peptide No. 222 RF-Pen-TGH-(3-Cl-Phe)-GGLYPC

3.7 Peptide No. 223 RF-Pen-TGH-(4-Cl-Phe)-GGLYPC

43 Peptide No. 224 RF-Pen-TGH-(3,3-Di-Phe)-GGLYPC

32 Peptide No. 225 RF-Pen-TGH-(4,4-Bi-Phe)-GGLYPC

>125 Peptide No. 226 RF-Pen-TGH-(4-t-Butyl-Phe)-GGLYPC

>125 Peptide No. 267 RF-Pen-TGH-((D/L)-betamethylPhe)-G-Sar-NMeLeu-YPC

16 *“Sar” = sarcosine; “NMeLeu” = N-methyl leucine ¹SYN927 is cyclizedvia an amide bond between the Asp and Lys side chains

Table 13 provides a listing of various peptides and peptide analogs, inwhich single amino acids have been substituted with tyrosine. The effectof the substitutions on the binding parameters of these peptides withhuman FcRn is also provided. Column 1 contains the peptide identifier.Column 2 contains the amino acid sequence of the peptides. Column 3shows the chemical structure of Tyr analog side-chain. Column 4 containsthe IC₅₀ of each peptide as determined by the IgG competition ELISAoutlined in Example 4. Columns 5 and 6 contain the K_(D) of each peptideas determined at pH 6 and pH 7.4, respectively, by the Biacore analysisoutlined in Example 6.

TABLE 13 Tyrosine Substitutions K_(D) K_(D) Sequence Tyr Analog IC₅₀ (pH6) pH 7.4 (SEQ ID NOS 1 & 146-155) Side Chain μM μM μM SEQ ID NO: 1QRFCTGHFGGL-Y-PCNGP

26 5.1 30 Peptide No. 26 QRFCTGHFGGL-F-PCNGP

>125 230 Peptide No. 32 QRF-Pen-TGHFGGL-Y-PCNGP

2 0.25 1.2 Peptide No. 63 QRF-Pen-TGHFGGL-(4-amino-Phe)-PCNGP

110 34 Peptide No. 64 QRF-Pen-TGHFGGL-(4-methoxyPhe)-PCNGP

120 31 Peptide No. 65 QRF-Pen-TGHFGGL-(pentafluoroPhe)-PCNGP

>125 72 Peptide No. 66 QRF-Pen-TGHFGGL-(2-pyridylAla)-PCNGP

>125 120 Peptide No. 67 QRF-Pen-TGHFGGL-(3-pyridylAla)-PCNGP

92 34 Peptide No. 68 QRF-Pen-TGHFGGL-(4-nitro-Phe)-PCNGP

122 180 Peptide No. 87 QRF-Pen-TGHFGGL-(2-nitro-Tyr)-PCNGP

>125 290 Peptide No. 140 QRF-Pen-TGHFGGL-(4-fluoro-Phe)-PCNGP

26 2.2 24

Table 14 provides a listing of Peptide No. 32-derived peptides andpeptide analogs, in which substitutions with various amino acid andamino acid derivatives have been generated where there is normally thesequence: Gly-Leu. Column 1 contains the peptide identifier. Column 2contains the amino acid sequence of the peptides. Column 3 contains theIC₅₀ of each peptide as determined by the IgG competition ELISA outlinedin Example 4. Columns 4 and 5 contain the K_(D) of each peptide asdetermined at pH 6 and pH 7.4, respectively, by the Biacore analysisoutlined in Example 6.

TABLE 14 Analogs of Peptide No. 32 at Gly-Leu K_(D) K_(D) Sequence IC₅₀(pH 6) pH 7.4 (SEQ ID NOS 156-164) μM μM μM Peptide No. 32QRF-Pen-TGHFGG-L-YPCNGP 2 0.25 1.2 Peptide No. 84QRF-Pen-TGHFGG-H-YPCNGP 6.5 0.38 2.5 Peptide No. 101QRF-Pen-TGHFGG-I-YPCNGP 3.4 0.34 Peptide No. 115 QRF-Pen-TGHFGG-F-YPCNGP4.1 0.40 Peptide No. 116 QRF-Pen-TGHFGG-W-YPCNGP 1.7 0.17Peptide No. 117 QRF-Pen-TGHFGG-M-YPCNGP 7.7 0.44 Peptide No. 118QRF-Pen-TGHFGG-L-YPCNGP 8.6 0.80 Peptide No. 237 RF-Pen-TGHFGG-W-YPC 2.80.14 Peptide No. 238 QRF-Pen-TGHFG-Sar-W-YPCNGP 1.0 0.068

Table 15 provides a listing of Peptide No. 32-derived peptides andpeptide analogs with a substitution of a glycine and a leucine takentogether for a dipeptide mimetic where there is normally the sequence:Gly-Leu. Column 1 contains the peptide identifier. Column 2 contains theamino acid sequence of the peptides. Column 3 provides the descriptionfor the identity of X in the sequence. Column 4 shows the chemicalstructure of the analog designated X. Column 5 contains the IC₅₀ of eachpeptide as determined by the IgG competition ELISA outlined in Example4. Column 6 contains the K_(D) of each peptide as determined at pH 6 bythe Biacore analysis outlined in Example 6.

TABLE 15 Peptidomimetic Analogs of Peptide No. 32 at Gly-Leu K_(D) ¹Sequence X X IC₅₀ (pH 6) (SEQ ID NOS 165-167) Description Structure μMμM Peptide No. 32 QRF-Pen-TGHFG-X-YPCNGP Gly-Gly-Leu

2.0 0.25 Peptide No. 216 QRF-Pen-TGHFG-X-YPCNGP L,L-Friedinger's lactam

19 Peptide No. 194 QRF-Pen-TGHFG-X-YPCNGP D,L-Friedinger's lactam

4.9

Example 8 Synthesis of Peptides Containing Histidine Analogs

Modified histidine analogs (Table 16) were synthesized as described inExample 7 for the synthesis of monomeric peptide disulfides except forthe following modified histidine analogs. Peptide No. 259 wassynthesized by suspending the resin containing the fully protectedpeptide analogous to Peptide No. 99 in neat methyl iodide for 15 hours.The resin washed with dichloromethane and the peptide was cleaved fromthe resin, oxidized and purified by HPLC as described above to yield themono-methylated histidine peptide Peptide No. 259.

Peptide No. 260 was synthesized by suspending the resin containing thefully protected peptide analogous to Peptide No. 99 in neat methyliodide for 72 hours. The resin washed with dichloromethane and thepeptide was cleaved from the resin, oxidized and purified by HPLC asdescribed above to yield the di-methylated histidine peptide Peptide No.260.

Peptide No. 269 was synthesized by suspending the resin containing thefully protected peptide analogous to Peptide No. 248 in dichloromethaneunder nitrogen. Ten molar equivalents of 2,4,6-tri-tert-butylpyridine(Sigma-Aldrich, St. Louis, Mo.) were added to the suspension followed byfive molar equivalents of methyl-trifluoromethane-sulfonate(Sigma-Aldrich, St. Louis, Mo.). The reaction was allowed to proceed for4 hours while rocking and rinsed first with dichloromethane, followed bya rinse with dimethylformamide and finally with dichloromethane again.The peptide was cleaved from the resin, oxidized and purified by HPLC asdescribed above to yield the N-methyl-thiazolium peptide, Peptide No.269.

Peptide No. 271 was synthesized by treating the peptide Peptide No. 261with 30 equivalents of copper sulfate, 30 equivalents of ascorbic acidand 10 equivalents of sodium azide in a solution of 100 mM sodiumphosphate buffer at pH 7.5 with 33% ethanol, 10% acetonitrile, 10%N,N-dimethylformamide. The reaction proceeded for 2 hours and themixture was purified by HPLC as described above to yield the1,2,3-triazole side-chain containing peptide Peptide No. 271.

Table 16 provides a listing of various peptides and peptide analogs, inwhich single amino acids have been substituted for histidine. The effectof the substitutions on the binding parameters of these peptides withhuman FcRn is also provided. Column 1 contains the peptide identifier.Column 2 contains the amino acid sequence of the peptides. Column 3shows the chemical structure of H is analog side-chain. Column 4contains the IC₅₀ of each peptide as determined by the IgG competitionELISA outlined in Example 4. Columns 5 and 6 contain the K_(D) of eachpeptide as determined at pH 6 and pH 7.4, respectively, by the Biacoreanalysis outlines in Example 6.

TABLE 16 Histidine Substitutions K_(D) K_(D) Sequence His Analog IC₅₀(pH 6) pH 7.4 (SEQ ID NOS 1 & 168-196) Side Chain μM μM μM SEQ ID NO: 1QRFCTG-H-FGGLYPCNGP

26 5.1 30 Peptide No. 36 QRFCTG-Dab-FGGLYPCNGP

>125 211 Peptide No. 32 QRF-Pen-TG-H-FGGLYP-C-NGP

2 0.25 1.2 Peptide No. 91 QRF-Pen-TG-Thz-FGGLYPCNGP

44 7.9 21 Peptide No. 109 QRF-Pen-TG-Dap-FGGLYPCNGP

>125 >100 Peptide No. 297 QRF-Pen-TG-Dap(Guanyl)-FGGLYPCNGP

54 13 16 Peptide No. 138 QRF-Pen-TG-(1Me)His-FGGLYPCNGP

3.4 0.74 14 Peptide No. 139 QRF-Pen-TG-Dab-FGGLYPCNGP

64 7.3 8.4 Peptide No. 192 RF-Pen-TG-NMeHis-FGGLYPC

>250 nd nd Peptide No. 248 RF-Pen-TG-Thz-FG-Sar-NMeL-YPC

1.6 .84 1.1 Peptide No. 249 RF-Pen-TG-2PyridylAla-FG-Sar-NMeL-YPC

6.2 .33 0.41 Peptide No. 250 RF-Pen-TG-3PyridylAla-FG-Sar-NMeL-YPC

1.2 .064 0.26 Peptide No. 251 RF-Pen-TG-ThienylAla-FG-Sar-NMeL-YPC

45 2 3 Peptide No. 253 RF-Pen-TG-Dab-FG-Sar-NMeL-YPC

16 1.2 1.2 Peptide No. 254 RF-Pen-TG-Orn-FG-Sar-NMeL-YPC

12 1.3 1.2 Peptide No. 255 RF-Pen-TG-Lys-FG-Sar-NMeL-YPC

40 1.3 1.1 Peptide No. 256 RF-Pen-TG-Arg-FG-Sar-NMeL-YPC

5.5 0.5 0.5 Peptide No. 257 RF-Pen-TG-4GuanylPhe-FG-Sar-NMeL-YPC

1.7 0.074 0.073 Peptide No. 258 RF-Pen-TG-4aminoPhe-FG-Sar-NMeL-YPC

4.6 0.22 1.1 Peptide No. 259 RF-Pen-TG-His(Me)-FGGLYPC

2.9 0.14 0.38 Peptide No. 260 RF-Pen-TG-His(Me)2-FGGLYPC

4.4 0.19 0.46 Peptide No. 261 RF-Pen-TG-PropargylGly-FG-Sar-NMeLeu-YPC

160 13 11 Peptide No. 262RF-Pen-TG-(2-PyrrolidinylAla)-FG-Sar-NMeLeu-YPC

150 8.4 13 Peptide No. 263 RF-Pen-TG-(3-PiperidyalAla)-FG-Sar-NMeLeu-YPC

6.3 0.66 0.86 Peptide No. 264RF-Pen-TG-(4-PiperidylAla)-FG-Sar-NMeLeu-YPC

85 5.2 6.4 Peptide No. 265 RF-Pen-TGFFG-Sar-NMeLeu-YPC

27 3.3 4.2 Peptide No. 266 RF-Pen-TGAFG-Sar-NMeLeu-YPC

>100 9.9 13 Peptide No. 268 RF-Pen-TG-(4-PyridylAla)-FG-Sar-NMeLeu-YPC

1.3 0.067 0.28 Peptide No. 269 RF-Pen-TG-Thz(Me)-FG-Sar-NMeL-YPC-CONH2

2.4 0.11 0.11 Peptide No. 271 RF-Pen-TG-triazolylAla-FG-Sar-NMeL-YPC

5.7 0.32 0.36

Example 9 Synthesis of Peptides Containing Peptidomimetic Analogs ofGly-Gly

All of the Gly-Gly amino acid mimetics (Table 17) were incorporated astheir Fmoc-amino protected amino acids and were commercially availableunless otherwise noted (Chem-Impex, Wood Dale, Ill.). Peptidescontaining 3(R)-3-amino-2-oxo-1-piperidine-acetic were synthesized byincorporating the N-Fmoc derivative of3(R)-3-amino-2-oxo-1-piperidine-acetic acid into Peptide No. 227according to the protocol described by R. M. Freidinger et. al., J. Org.Chem. 47: 104-109 (1982). Peptides containing3(R)-3-amino-2-oxo-1-pyrrolidine acetic acid were synthesized byincorporating the N-Fmoc derivative of 3(R)-3-amino-2-oxo-1-pyrrolidineacetic acid into Peptide No. 214 according to the protocol described byR. M. Freidinger et. al., J. Org. Chem. 47:104-109 (1982). Peptidescontaining the 5,5-bicyclic dipeptide mimic were synthesized byincorporating the 5,5-bicyclic dipeptide mimic into Peptide No. 197 orPeptide No. 198 according to the protocol described by N. L. Subasingheet. al., J. Med. Chem. 36: 2356-2361 (1993) with the exception that allD-amino acids were used. Peptides containing the 6,5-bicyclic dipeptidemimic were synthesized by incorporating the 6,5-bicyclic dipeptide mimicinto Peptide No. 204 according to the protocol described by F. A.Etzkorn et. al., J. Am. Chem. Soc. 116: 10412 (1994) with the exceptionthat all D-amino acids were used. Peptides containing the(D,L)-Freidinger's lactam were synthesized by incorporating the(D,L)-Freidinger's lactam into Peptide No. 216 according to the protocoldescribed by R. M. Freidinger et al., J. Org. Chem. 47: 104-109 (1982)with the exception that L-methionine was used instead of D-methionine.

Table 17 provides a listing of SEQ ID NO:1-derived peptides and peptideanalogs in which substitutions with various amino acid and amino acidderivatives have been generated where there are normally two adjacentglycines (Gly-Gly). Column 1 contains the peptide identifier. Column 2contains the amino acid sequence of the peptides. Column 3 contains theIC₅₀ of each peptide as determined by the IgG competition ELISA outlinedin Example 4. Columns 4 and 5 contain the K_(D) of each peptide asdetermined at pH 6 and pH 7.4, respectively, by the Biacore analysisoutlined in Example 6.

TABLE 17 Analogs of SEQ ID NO:1 at Gly-Gly K_(D) K_(D) Sequence IC₅₀(pH 6) pH 7.4 (SEQ ID NOS 197-201) μM μM μM SEQ ID NO:1QRFCTGHFGGLYPCNGP 26 5.1 30 Peptide No. 22 QRFCTGHF-a-GLYPCNGP 48 10 137Peptide No. 23 QRFCTGHFG-a-LYPCNGP 57 12 184 Peptide No. 24QRFCTGHF-a-a-LYPCNGP 69 22 >250 Peptide No. 25QRFCGHF-betaAla-LYPCNGP >125 >250 nd Peptide No. 35QRFCTGHF-Apa-LYPCNGP >125 220 nd * “beta-Ala” = beta-alanine; “Apa” =5-aminopentanoic acid

Table 18 provides a listing of Peptide No. 99-derived peptides andpeptide analogs with a substitution of two glycines taken together for apeptidomimetic analog where there is normally the sequence: Gly-Gly.Column 1 contains the peptide identifier. Column 2 contains the aminoacid sequence of the peptides. Column 3 provides the description for theidentity of the peptidomimetic analog designated as X in the sequence.Column 4 shows the chemical structure of the analog designated X. Column5 contains the IC₅₀ of each peptide as determined by the IgG competitionELISA outlined in Example 4. Column 6 contains the K_(D) of each peptideas determined at pH 6 by the Biacore analysis outlined in Example 6.

TABLE 18 Peptidomimetic Analogs of Peptide No. 99 at Gly-Gly K_(D)Sequence X X IC₅₀ (pH 6) (SEQ ID NOS 202-219) Description Structure μMμM Peptide No. 99 RF-Pen-TGHF-X-LYPC Gly-Gly

2.0 0.17 Peptide No. 134 RF-Pen-TGHF-X-LYPC 4-aminomethyl-benzoic acid

>125 Peptide No. 135 RF-Pen-TGHF-X-LYPC (3-aminomethyl)-benzoic acid

57 Peptide No. 136 RF-Pen-TGHF-X-LYPC 4-aminophenyl acetic acid

>125 Peptide No. 137 RF-Pen-TGHF-X-LYPC 3-aminophenyl acetic acid

14 Peptide No. 178 RF-Pen-TGHF-X-LYPC 3-amino-2-oxo-1-piperidine-aceticacid

0.66 0.16 Peptide No. 179 RF-Pen-TGHF-X-LYPC3-amino-2-oxo-1-piperidine-acetic acid

7.2 0.67 Peptide No. 193 RF-Pen-TGHF-X-LYPC3(S)-3-amino-2-oxo-1-piperidine-acetic acid

7.3 Peptide No. 80 RF-Pen-TGHF-X-LYPC 3-amino-N-1-carboxymethyl-2,3,4,5-tetrahydro-1H-[1]-benzazepine-2-one

159 Peptide No. 181 RF-Pen-TGHF-X-LYPC3-amino-N-1-carboxymethyl-2,3,4,5- tetrahydro-1H-[1]-benzazepine-2-one

1.2 Peptide No. 197 RF-Pen-TGHF-X-LYPC 5,5-bicyclic dipeptide mimic

13 0.99 Peptide No. 198 RF-Pen-TGHF-X-LYPC 5,5-bicyclic dipeptide mimic

23 Peptide No. 204 RF-Pen-TGHF-X-LYPC 6,5-bicyclic dipeptide mimic

2.2 0.32 Peptide No. 205 RF-Pen-TGHF-X-LYPC 3(R)-3-amino-2-oxo-1-azepineacetic acid

0.64 0.103 Peptide No. 214 RF-Pen-TGHF-X-LYPC3(R)-3-amino-2-oxo-1-pyrrolidine acetic acid

2.3 0.28 Peptide No. 227 RF-Pen-TGHF-X- NMeLeu-YPC3(R)-3-amino-2-oxo-1-piperidine-acetic acid

0.53 0.043 Peptide No. 228 RF-Pen-NMeAla-GHF- X-NMeLeu-YPC3(R)-3-amino-2-oxo-1-piperidine-acetic acid

1.1 0.145 Peptide No. 239 RF-Pen-TGHF-X- NMeLeu-YPC3(R)-3-amino-2-oxo-1-azepine acetic acid

0.62 0.044

Example 10 Synthesis of Peptides Cyclized Via A Lactam Bridge

Lactam cyclized peptides (Table 19) were synthesized by solid-phasepeptide synthesis as outlined above in Example 7 with the exception thatthe following amino acids were used as substitutes for variouscysteines: Fmoc-Lys(Aloc)-OH, Fmoc-Orn(Aloc)-OH, Fmoc-Dab(Aloc)-OH andFmoc-Dap(Aloc)-OH, Fmoc-Glu(OAllyl)-OH and Fmoc-Asp(OAllyl)-OH (Bachem,Torrance, Calif.). Following the completion of the process to generatefully protected peptides on resin, the resin was swollen indichloromethane, purged with nitrogen and treated with 0.1 molarequivalents of tetrakis-(triphenylphosphine)palladium(0) (Sigma-Aldrich,St. Louis, Mo.) and 30 molar equivalents of phenylsilane (Sigma-Aldrich,St. Louis, Mo.) and the reaction was allowed to proceed for three hours.The resin washed first with dichloromethane, with DMF and finally fiveadditional times with a solution of 1% (v/v) triethylamine and 1% (w/v)diethyldithiocarbamic acid in DMF. An additional washing step with DMFwas followed by treatment of the resin withbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) (Novabiochem, San Diego Calif.) and DIEA for 16 hours. Thepeptides were cleaved from the resin and purified as described above inExample 7.

Table 19 provides a listing of various peptides of the invention withamino acid substitutions of cysteine residues for amino acids and aminoacid analogs that would allow for the cyclization of the respectivepeptides via a lactam bridge. The impact of the substitutions on thebinding parameters of these peptides with human FcRn is also provided.Column 1 contains the peptide identifier. Column 2 contains the aminoacid sequence of the peptides. Column 3 contains the IC₅₀ of eachpeptide as determined by the IgG competition ELISA outlined in Example4. Columns 4 and 5 contain the K_(D) of each peptide as determined at pH6 and pH 7.4, respectively, by the Biacore analysis outlined in Example6.

TABLE 19 Lactam Cyclized Peptides K_(D) K_(D) Sequence¹ IC₅₀ (pH 6)pH 7.4 (SEQ ID NOS 220-246) μM μM μM Peptide No. 38QRF-Asp-TGHFGGLYP-Dab-NGP 68 17 150 Peptide No. 39QRF-Asp-TGHFGGLYP-Lys-NGP 10 1.2 12 Peptide No. 72QRF-Dab-TGHFGGLYP-Glu-NGP 81 4.8 nd Peptide No. 73QRF-Lys-TGHFGGLYP-Glu-NGP 33 1.2 nd Peptide No. 76QRF-Glu-TGHFGGLYP-Lys-NGP 100 27 320 Peptide No. 77QRF-Glu-TGHFGGLYP-Dab-NGP 86 22 210 Peptide No. 78QRF-Glu-TGHFGGLYP-Dap-NGP 71 9.6 81 Peptide No. 79QRF-Asp-TGHFGGLYP-Dap-NGP 32 4.5 30 Peptide No. 80QRF-Lys-TGHFGGLYP-Asp-NGP 60 10 52 Peptide No. 81QRF-Dab-TGHFGGLYP-Asp-NGP 31 8.4 45 Peptide No. 85QRF-Asp-TGHFGGLYP-Orn-NGP 16 2.7 nd Peptide No. 86QRF-Glu-TGHFGGLYP-Orn-NGP 80 15 nd Peptide No. 107QRF-Asp-TGHFGGLY-Lys-NGP >125 >250 nd Peptide No. 105QRF-Asp-TGHFG-a-LYP-Lys-NGP 12 2.1 nd Peptide No. 106QRF-Asp-TGHF-a-GLYP-Lys-NGP 17 3.7 nd Peptide Np. 123Asp-TGHFGGLYP-Lys-NGP 47 Peptide No. 124 F-Asp-TGHFGGLYP-Lys-NGP 22Peptide No. 125 RF-Asp-TGHFGGLYP-Lys-NGP 9.4 Peptide No. 126QRF-Asp-TGHFGGLYP-Lys-NGP 13 Peptide No. 127 QRF-Asp-TGHFGGLYP-Lys-N 7.6Peptide No. 128 QRF-Dap-TGHFGGLYP-Asp-NGP 120 Peptide No. 129QRF-Dap-TGHFGGLYP-Glu-NGP >125 Peptide No. 130 QRF-Orn-TGHFGGLYP-Asp-NGP120 Peptide No. 131 QRF-Orn-TGHFGGLYP-Glu-NGP 30 Peptide No. 132RF-Asp-TGHFGGLYP-Lys 11 0.90 Peptide No. 133 QRF-Asp-TGHFGGLYP-Lys 130.90 Peptide No. 159 QRF-Asp-TGHFG-p-LYP-Lys-NGP 15 1.2 ¹ There is anamide bond between the side-chains of the underlined amino acids; Dab =1,3-diaminobutyric acid; Dap = 1,2-diaminoproprionic acid; Orn =ornithine

Example 11 Synthesis of Linear Peptide Analogs

Linear peptide analogs were synthesized as described above in Example 7,with the exception that disulfide-forming amino acids were substitutedas set forth in Tables 20 and 21.

Table 20 provides a listing of SEQ ID NO:1-derived linear peptides andpeptide analogs of the invention. The binding parameters of thesepeptides with human FcRn is also provided. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6.

TABLE 20 Linear Analogs of SEQ ID NO:1 K_(D) K_(D) Sequence¹ IC₅₀ (pH 6)pH 7.4 (SEQ ID NOS 1 & 247-273) μM μM μM SEQ ID NO:1 QRFCTGHFGGLYPCNGP26 5.1 30 Peptide No. 71 QRF-S-TGHFGGLYP-S-NGP >125 230 Peptide No. 156QRF-V-TGHF-p-p-LYP-A-NGP >250 Peptide No. 157 QRF-V-TGHF-G-p-LYP-A-NGP195 16 Peptide No. 58 QRF-V-TGHF-p-G-LYP-A-NGP >250 Peptide No. 162QRF-L-TGHF-G-p-LYP-A-NGP >250 Peptide No. 163QRF-I-TGHF-G-p-LYP-A-NGP >250 Peptide No. 164QRF-F-TGHF-G-p-LYP-A-NGP >250 Peptide No. 165QRF-Y-TGHF-G-p-LYP-A-NGP >250 Peptide No. 166QRF-W-TGHF-G-p-LYP-A-NGP >250 Peptide No. 167 QRF-V-TGHF-G-p-LYP-A-NGP93 Peptide No. 168 QRF-V-TGHF-G-p-LYP-L-NGP 100 Peptide No. 169QRF-V-TGHF-G-p-LYP-I-NGP 72 15 Peptide No. 170QRF-V-TGHF-G-p-LYP-F-NGP >250 Peptide No. 171 QRF-V-TGHF-G-p-LYP-Y-NGP150 Peptide No. 172 QRF-V-TGHF-G-p-LYP-W-NGP 150 Peptide No. 173QRF-V-TGHF-G-p-V-YP-A-NGP >250 Peptide No. 174 QRF-V-TGHF-G-p-I-YP-A-NGP94 Peptide No. 175 QRF-V-TGHF-G-p-F-YP-A-NGP 200 Peptide No. 176QRF-V-TGHF-G-p-Y-YP-A-NGP 230 Peptide No. 177 QRF-V-TGHF-G-p-W-YP-A-NGP52 5.8 96 Peptide No. 190 QRF-V-TGHF-G-p-W-YP-I-NGP 49 4.2Peptide No. 209 RF-V-TGHF-G-p-W-YP >125 Peptide No. 210RF-V-TGHF-G-p-W-YP-A-NGP 100 10 Peptide No. 211 F-V-TGHF-G-p-W-YPA 100 8Peptide No. 212 V-TGHF-G-p-W-YP-A >250 Peptide No. 236RF-V-TGHF-G-Sar-NMeLeu-YP-A 37 1.85 9 Peptide No. 246RF-V-TGHF-G-p-W-YPA 60 3.6 ¹“Sar” = sarcosine; “NMeLeu” = N-methylleucine

Table 21 provides a listing of Peptide No. 236-derived peptides wherevarious peptidomimetic analogs have been substituted where there isnormally a Glycine-Sarcosine sequence (Gly-Sar). Column 1 contains thepeptide identifier. Column 2 contains the amino acid sequence of thepeptides. Column 3 provides the description for the identity of thepeptidomimetic analog designated as X in the sequence. Column 4 showsthe chemical structure of the peptidomimetic analog designated X. Column5 contains the IC₅₀ of each peptide as determined by the IgG competitionELISA outlined in Example 4. Column 6 contains the K_(D) of each peptideas determined at pH 6 by the Biacore analysis outlined in Example 6.

TABLE 21 Linear Analogs of Peptide No. 236 with Gly-Gly peptidomimeticsK_(D) Sequence X X IC₅₀ (pH 6) (SEQ ID NOS 274-283) DescriptionStructure μM μM Peptide No. 236 RF-V-TGHF-X-NMeLeu-YPA Gly-Sar

37 1.85 Peptide No. 182 RF-V-TGHF-X-LYPA3-amino-2-oxo-1-piperidine-acetic acid

38 3.3 Peptide No. 183 RF-V-TGHF-X-LYPA3-amino-2-oxo-1-piperidine-acetic acid

>250 Peptide No. 184 RF-V-TGH F-X-LYPA3-amino-N-1-carboxymethyl-2,3,4,5- tetrahydro-1H-[1]-benzazepine-2-one

>250 Peptide No. 185 RF-V-TGH F-X-LYPA3-amino-N-1-carboxymethyl-2,3,4,5- tetrahydro-1H-[1]-benzazepine-2-one

57 3.1 Peptide No. 186 RF-V-TGHF-X-LYPA 3-aminophenyl acetic acid

>250 Peptide No. 191 QRF-V-TGHF-X-WYPINGP3-amino-2-oxo-1-piperidine-acetic acid

nd 333 Peptide No. 206 RF-V-TGHF-X-LYPA 5,5-bicyclic dipeptide mimic

>250 Peptide No. 207 RF-V-TGHF-X-LYPA 6,5-bicyclic dipeptide mimic

>125 20 Peptide No. 208 RF-V-TGHF-X-LYPA 3(R)-3-amino-2-oxo-1-azepineacetic acid

23 2.3 *“Sar” = sarcosine; “NMeLeu” = N-methyl leucine

Example 12 Synthesis of Peptide Dimers Via Reductive Alkylation

Peptide dimers (Table 22) were generated by reductive alkylation of apeptide aldehyde and a peptide amino (N) or carboxy (C) terminal amine.

Peptide N-terminal amines were synthesized as described above in Example7 for the synthesis of monomeric peptide disulfides.

Peptide C-terminal amines were also synthesized as described above inExample 7 for the synthesis of monomeric peptide disulfides, except that1,2-diaminoethane resin (Novabiochem, San Diego, Calif.) was used in thesynthesis step. Consequently, cleavage from the resin resulted in aC-terminal ethyl amine.

Peptide N-terminal aldehydes (FIG. 2) were synthesized by reacting theunprotected amine of the N-terminal amino acid with 5 equivalents ofsuccinnic anhydride (Sigma-Aldrich, St. Louis, Mo.) in the presence ofDIEA in DMF for 2 hours. A subsequent reaction with2,2-dimethyl-1,3-dioxolane methamine (Sigma-Aldrich, St. Louis, Mo.) inthe presence of PyBOP and DIEA for 2 hours yielded the protected diolresin. Then, cleavage of the crude peptide from the resin, followed bycysteine oxidation and purification as described above in Example 7 forthe synthesis of monomeric peptide disulfides, yielded the peptide diol.The diol was dissolved in 33% acetic acid followed by 2 equivalents ofsodium periodate (Sigma-Aldrich, St. Louis, Mo.) was added and thereaction was allowed to proceed for 5 minutes. The reaction mixture wasquenched with 20 equivalents (with respect to the diol) of ethyleneglycol (Sigma-Aldrich, St. Louis, Mo.) and after ten minutes, the crudereaction mixture was diluted 3-fold with water and purified over a C18Sep-Pak column (Waters Corp., Milford, Mass.) using an increasinggradient of acetonitrile in water containing 0.1% TFA. The peptidealdehyde was lyophilized and subjected to analysis by mass spectroscopy(Mariner ES-MS) following liquid chromatography (Applied Biosystems,Foster City, Calif.) as described in Example 7.

Peptide C-terminal aldehydes were synthesized as described above inExample 7 for the synthesis of monomeric peptide disulfides, except thatFmoc-1-amino-2,3-propanediol-2′-chlorotrityl resin (Novabiochem, SanDiego, Calif.) was used instead of Rink amide resin. Therefore theresulting peptide resin contained a masked C-terminal diol. Uponcleavage from the resin, the diol was oxidized to an aldehyde asdescribed above for N-terminal aldehydes.

Peptide monomers to synthesize lactam-cyclized peptides such as PeptideNo. 275, were synthesized according to the method described above inExample 10 to synthesize peptides cyclized by a lactam bridge, wherebythe Asp-Lys cyclization was performed on the resin, prior to cleavagefrom the resin.

The peptide dimers were synthesized (FIG. 3) by reacting one equivalentof peptide aldehyde with one equivalent of amine-containing peptide at aconcentration of 40 mg/ml in DMF containing 2% acetic acid. After 60min., 2 equivalents of sodium cyanoborohydride (Sigma-Aldrich, St.Louis, Mo.) were added and the reaction was allowed to shake for 1 hour.The reaction mixture was diluted 10-fold with water and purified by HPLCand analyzed by mass spectroscopy (Mariner ES-MS) following liquidchromatography (Applied Biosystems, Foster City, Calif.) as described inExample 7.

Table 22 provides a listing of dimeric peptides of the invention thatwere synthesized by reductive alkylation. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6. Column 6 contains the IC₅₀of each peptide as determined by competitive IgG binding FACS analysisas outlined in Example 5.

TABLE 22 Dimers and Trimers Synthesized by Reductive Alkylation K_(D)K_(D) pH pH IC₅₀ IC₅₀ 6 7.4 nM Sequence¹ nM nM nM FACS Pep- tide No. 276

3700 56 12,900 Pep- tide No. 215

30 6.6 Pep- tide No. 230

7.2 <0.5 0.46 Pep- tide No. 231

30 2.9 Pep- tide No. 247

6 Pep- tide No. 270

2.6 <0.5 <0.8 4 Pep- tide No. 272

2.8 <0.5 <0.8 5 Pep- tide No. 273

2.1 <0.5 <0.9 4 Pep- tide No. 274

17 Pep- tide No. 277

6.3 Pep- tide No. 278

4.4 Pep- tide No. 275

44 1.6 9.1 X = 3(R)-3-amino-2-oxo-1-piperidine-acetic acid

Example 13 Synthesis of Peptide Dimers by Thiol Linkers andBromoacetylated Peptides

Peptide dimers (Table 23) were also synthesized by reactingbromoacetylated peptides with a thiol linkers. Bromoacetylated peptideswere synthesized (FIG. 4) by reacting the free α-amino group of theprotected peptide resin with 4 equivalents of bromoacetyl bromide(Sigma-Aldrich, St. Louis, Mo.) and 8 equivalents of DIEA(Sigma-Aldrich, St. Louis, Mo.) in DMF. After 1 hour, the resin waswashed with DMF, followed by DCM and cleaved from the resin as describedabove in Example 7. In the case where lactam-cyclized peptides weredimerized using a bis-thiol linker, the on-resin cyclization step wasperformed prior to the bromoacetylation step. In the case wheredisulfide-containing peptides were dimerized using a bis-thiol linker,the iodine oxidation step was performed after cleavage as describedabove in Example 7.

The bis-thiol linkers were synthesized (FIG. 4) by reactingNH₂-Gly-2-Chlorotrityl resin (Novabiochem, San Diego, Calif.) with 2equivalents of N,N-bis(N′-Fmoc-3-aminopropyl)glycine potassiumhemisulphate (Chem-Impex, Wood Dale, Ill.) in the presence of 2equivalents of PyBOP (Novabiochem, San Diego, Calif.) and DIEA in DMFfor 18 hours. The Fmoc protecting group was removed with two 10 minutetreatments of 20% piperidine in DMF. For some of the linker compounds,beta-alanines were also incorporated as spacer units. Fmoc-beta-Ala-OH(Novabiochem) was coupled to the resin as above using PyBOP and DIEA.After the Fmoc protecting group was removed with 20% piperidine in DMF,either another beta-alanine spacer unit was incorporated, or thebis-thiol linker was incorporated by reacting the free N-terminal amineresin with 2 equivalents of N-succinimidyl-S-acetylthioproprionate(SATP; Pierce, Rockford, Ill.) and 4 equivalents of DIEA for 18 hours.

Subsequently, removal of the S-acetyl protecting group was accomplishedby reacting 0.05 mmol of the peptide resins with a degassed solutioncontaining 1 ml of DMF and 0.4 ml of buffer A (Buffer A: 1 Mhydroxylamine hydrochloride (Sigma-Aldrich, St. Louis, Mo.), 40 mMsodium phosphate pH 7.5, 50 mM EDTA (Sigma-Aldrich, St. Louis, Mo.)) for18 hours. The resins were washed with DMF, followed by DCM, and cleavedfrom the resin with a 50% solution of TFA in DCM with 2%triisopropylsilane for 15 min. The crude linkers were processed andpurified as described above in Example 7.

The peptide dimers were generated using bis-thiol linkers (FIG. 4) byreacting one equivalent of the purified bis-thiol linker with a twoequivalents of bromoacetylated N-terminal peptide in DMF with 10% waterand 50% 100 mM sodium phosphate, pH 7.5. After 18 hours, the crudereaction mixture was purified by reversed phase HPLC column as describedabove in Example 7.

Peptide No. 122 was synthesized (FIG. 5) by reacting a bromoacetylatedpeptide with a peptide derivatized with SATP. Briefly, the crude peptideresin with a free N-terminal amine was reacted with 2 equivalents ofSATP in DMF for 2 hours. The S-acetyl protecting group was removed asdescribed above, followed by cleavage from the resin and subsequentpurification as described above.

Table 23 provides a listing of dimeric peptides of the invention thatwere synthesized by thiol linkers. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4. Columns 4 and 5 contain theK_(D) of each peptide as determined at pH 6 and pH 7.4, respectively, bythe Biacore analysis outlined in Example 6. Column 6 contains the IC₅₀of each peptide as determined by competitive IgG binding FACS analysisas outlined in Example 5.

TABLE 23 Dimers Synthesized Using Thiol Linkers Sequence¹ Peptide No.100

Peptide No. 119

Peptide No. 120

Peptide No. 121

Peptide No. 122

Peptide No. 160

Peptide No. 161

Peptide No. 199

Peptide No. 200

IC₅₀ nM IC₅₀ nM K_(D) pH 6 nM K_(D) pH 7.4 nM FACS Peptide No. 100 760 6130 Peptide No. 119 900 7 150 Peptide No. 120 2400 7 150 Peptide No. 1211300 8 160 Peptide No. 122 970 6 120 Peptide No. 160 100 Peptide No. 16190 Peptide No. 199 1200 7.9 190 Peptide No. 200 1900 7.2 170 ¹Pen =penicillamine; Sar = sarcosine; p = D-proline; NMeLeu = N-methylleucine

Example 14 Synthesis of Peptide Trimers Via Reductive Alkylation:Peptide No. 247

Peptide trimers (Table 22) were generated by reductive alkylation of apeptide aldehyde and a peptide amino N-terminal amine.

Peptide N-terminal amines were synthesized as described above in Example7 for the synthesis of monomeric peptide disulfides with the exceptionthat the N-terminus was capped with a bifunctional amine linker such asbis-aminipropyl glycine (BAPG; used as Bis-Fmoc-BAPG purchased fromSigma-Aldrich, St. Louis, Mo.), followed by coupling sarcosine. PeptideN-terminal aldehydes (FIG. 2) were synthesized as described in Example12. The peptide trimers were synthesized (as in FIG. 3) by reacting twoequivalents of peptide aldehyde with one equivalent of amine-containingpeptide at a concentration of 40 mg/ml in DMF containing 2% acetic acid.After 60 min., 4 equivalents of sodium cyanoborohydride (Sigma-Aldrich,St. Louis, Mo.) were added and the reaction was allowed to shake for 1hour. The reaction mixture was diluted 10-fold with water and purifiedby HPLC and analyzed by mass spectroscopy (Mariner ES-MS) followingliquid chromatography (Applied Biosystems, Foster City, Calif.) asdescribed in Example 7.

Example 15 Synthesis Of Peptide Dimers Using Diacid and Amine Linkers

Amide linked peptide dimers (Table 24) were generated either by reactingthe N-termini of two on-resin peptide monomers with a bi-functional acidlinker or by performing the synthesis of the peptide on resin containinga bi-functional amine linker, thereby tethering the C-termini of twoon-resin peptide monomers.

N-terminally linked peptide dimers were synthesized as described abovein Example 7 for the synthesis of monomeric peptide disulfides with thefollowing exceptions: Before the peptides are cleaved from the resin,the N-termini of two peptide monomers are joined with a bi-functionalacid linker. For example, Peptide No. 283 is synthesized by reacting thepeptide resin containing the peptide sequence analogous to Peptide No.235 with an unprotected N-terminus with 0.5 equivalents of succinic acid(Sigma-Aldrich, St. Louis, Mo.) in the presence of 1 equivalent of PyBOPand 2 equivalents of DIEA. This results in adjacent peptides on theresin being covalently attached by amide bonds via their N-termini.

The resulting peptide dimer is cleaved from the resin and purified asdescribed in Example 7 with the exception that the peptide disulfidesare not oxidized prior to HPLC purification. The purified reducedpeptide is dissolved to ca. 0.1 mg/mL in 10 mM sodium phosphate, pH 7.5with 20% DMSO and mixed for 3 days at room temperature. This oxidationstep permits the formation of the disulfide bonds within one peptidemonomer of the dimer, as opposed to between two monomers of a dimer. Thereaction mixture is diluted with water to peptide concentration of 0.05mg/mL and purified over a C18 Sep-Pak column (Waters Corp., Milford,Mass.) using an increasing gradient of acetonitrile in water containing0.1% TFA. The peptide dimer was lyophilized and subjected to analysis bymass spectroscopy (Mariner ES-MS) following liquid chromatography(Applied Biosystems, Foster City, Calif.) as described in Example 7.(See FIG. 6.) In the case of Peptide No. 283, the disulfide linkagepattern was confirmed by digesting the peptide with trypsin for 30minutes, then analyzing the resulting peptides by LCMS. Trypsin is knownto cleave after arginine and lysine residues, and cleaves Peptide No.283 at the arginine-phenylalanine bond. The major product of LCMS ofPeptide No. 283 is NH2-[PhePhe-Pen-Thr-Gly-His-Phe-Gly-Sar-NMeLeu-Tyr-Pro-Cys]-CONH2(disulfide)(LCMS: M+H=1355.6 Da), which indicates that the disulfide bonds ofPeptide No. 283 are formed between each 13 amino acid peptide monomer.

Peptide No. 201 was synthesized as Peptide No: 283 with the exceptionsthat the peptide sequence was analogous to Peptide No. 32, the diacidlinker used was ethylene glycol-bis(succinic acid-N-hydroxysuccinimideester) (Sigma-Aldrich, St. Louis, Mo.) and that no PyBOP was used forthe coupling reaction.

Peptide No. 279 was synthesized as in Peptide No. 283 with the exceptionthat the diacid linker used was Bis-dPEG6-N-hydroxysuccinimide ester(Quanta Biodesigns Ltd.) and that no PyBOP was used for the couplingreaction.

Peptide No. 281 was synthesized as Peptide No. 283 with the exceptionthat the peptide-resin was treated with a large excess of succinicanhydride (Sigma-Aldrich, St. Louis, Mo.), which results in all peptideson the resin containing a succinate capped N-terminus. This resin wastreated with 0.5 equivalents of N,N′-dimethylethyl-enediamine(Sigma-Aldrich, St. Louis, Mo.) in the presence of 1 equivalent of PyBOPand 2 equivalents of DIEA. The subsequent cleavage, purification andoxidation steps were performed as with Peptide No. 283.

Peptide No. 282 was synthesized as Peptide No. 283 with the exceptionthat the diacid linker used was N-methyl-iminodiacetic acid(Sigma-Aldrich, St. Louis, Mo.).

Peptide No. 284 was synthesized as Peptide No. 283 with the exceptionthat the diacid linker used was 3,3-dimethylglutaric acid(Sigma-Aldrich, St. Louis, Mo.).

Peptide No. 285 was synthesized as Peptide No. 283 with the exceptionthat the diacid linker used was Boc-Asp(OH)—OH (Novabiochem, San Diego,Calif.).

Peptide No. 286 was synthesized as Peptide No. 283 with the exceptionthat the diacid linker used was Boc-Glu(OH)—OH (Novabiochem, San Diego,Calif.).

C-terminally linked peptide dimers were synthesized as described abovein Example 7 for the synthesis of monomeric peptide disulfides with theexception that a bifunctional amine linker is coupled to the resin priorto the peptide synthesis. This results in peptide dimers with theirC-termini covalently attached by amide bonds. For example, Peptide No.280 was synthesized by first coupling Fmoc-Lys(Fmoc)-OH (Novabiochem,San Diego, Calif.) to the resin, followed by the coupling of amino acidsto give a sequence analogous to Peptide No. 235. This results in thecovalent attachment of two peptide chains as they are being synthesizedon the resin. The resulting peptide dimer is cleaved from the resin,purified and oxidized as described above for the N-terminally linkeddimers. (see FIG. 7)

Peptide No. 287 was synthesized as Peptide No. 280 with the exceptionthat a glycine residue (Gly) is inserted between the Peptide No. 235sequence and the branching Lysine linker.

Peptide No. 288 was synthesized as Peptide No. 280 with the exceptionsthat two glycine residues (Gly-Gly) are inserted between the Peptide No.235 sequence and the branching lysine linker.

Table 24 provides a listing of dimeric peptides of the invention thatwere synthesized using amide bonds. Column 1 contains the peptideidentifier. Column 2 contains the amino acid sequence of the peptides.Column 3 contains the IC₅₀ of each peptide as determined by the IgGcompetition ELISA outlined in Example 4.

TABLE 24 Dimers Synthesized Using Amide Linkers Sequence¹ IC₅₀ (SEQ IDNOS 305-315) nM Peptide No. 201

26 Peptide No. 279

7 Peptide No. 280

25 Peptide No. 281

5.2 Peptide No. 282

4.7 Peptide No. 283

3.3 Peptide No. 284

8.5 Peptide No. 285

4.6 Peptide No. 286

5.6 Peptide No. 287

20 Peptide No. 288

16 ¹Pen = penicillamine; Sar = sarcosine; NMeLeu = N-methylleucine

Example 16 Synthesis of Peptide-Fc Fusions Via Reductive Alkylation

Peptide N-terminal aldehydes Peptide No. 252, Peptide No. 229 andPeptide No. 232 (Table 25) were synthesized as described in Example 12.All three peptide-Fc fusions were generated using the same protocol(FIG. 8): CysFc (Fc domain possessing a N-terminal cysteine) and 4.5equivalents of peptide aldehyde were incubated on ice in 80 mM sodiumacetate pH 5.5 for 1 hour. Sodium cyanoborohydride was added to a finalconcentration of 20 mM and the reaction was incubated for 16 hours at 4°C. The reaction mixture was analyzed by SDS-PAGE to ensure the additionof predominantly a single peptide to the Fc protein. The protein mixturewas dialyzed twice with PBS and assayed for in vitro blocking activity(Table 25). In the case of Peptide No. 252-Fc, the protein was alsoevaluated in the TG32B mouse IgG catabolism model. (FIG. 15). Theproduction of CysFc can be performed as described in US PatentApplication Publication No. US 2005/0027109, where the disclosure of theproduction of CysFc is incorporated herein by reference.

Table 25 provides a listing of peptide-Fc fusion proteins of theinvention that were synthesized using CysFc and aldehyde-peptides.Column 1 contains the peptide-Fc fusion identifier. Column 2 containsthe amino acid sequence of the peptides. Column 3 contains the IC₅₀ ofeach peptide as determined by the IgG competition ELISA outlined inExample 4.

TABLE 25 Peptide-Fc Fusions ID₅₀ Sequence¹ nM CysFc 210 Peptide No.229-Fc

 2 Peptide No. 232-Fc

 3 Peptide No. 252-Fc

 39 ¹Pen = penicillamine; Sar = sarcosine; NMeLeu = N-methylleucine

Example 17 Transgenic Mice

Transgenic mice were obtained from Dr. Roopenian of The JacksonLaboratory in Bar Harbor, Me. The endogenous murine FcRn and β₂m geneswere inactivated by insertion of a foreign polynucleotide sequence byhomologous recombination and replaced transgenically with the human FcRnand the human β₂m genes (muFcRn (−/−), muβ₂m (−/−), +huFcRn, +huβ₂m).These mice are referred to by the strain name TG32B.

Example 18 Effect of Peptide No. 270 on Human IgG Catabolism in TG32BMice Using 5 mg/kg and 10 mg/kg

Adult TG32B mice were injected intravenously with 500 mg/kg of human IgG(MP Biomedicals, Irvine, Calif.) at t=0 hours (T₀). At 24, 48, 72, 96and 120 hours, the mice were injected intravenously with either 5 mg/kgor 10 mg/kg Peptide No. 270. Control injections were performed at eachtimepoint using the vehicle PBS with 15 mM sodium acetate, pH 5. Bloodsamples were taken prior to injections at all timepoints, as well as at168 hours. Serum was prepared and stored at −20° C. until an ELISA wasperformed (FIG. 9).

An IgG Fc domain-specific ELISA was used to detect the levels of humanIgG in the serum at each time point. Briefly, 30 μl of a 10 μg/ml stocksolution of goat anti-human IgG (Pierce, Rockford, Ill.) was dilutedwith 6 ml of 0.05 M sodium bicarbonate, pH 9.6 (Sigma-Aldrich, St.Louis, Mo.). A 96-well plate was coated with 50 μl/well of this solutionand incubated for 1 hour at 37° C. The coating solution was removed andwashed once with PBST (phosphate buffered saline with 0.05% Tween-20).Then 200 μl/well of a 2% bovine serum albumin (BSA) stock solution inPBS was added and the plate incubated for 1 hour at 37° C. The wellswere washed three times with PBST and a standard curve was generated intriplicate by performing 2.5-fold dilutions starting from 50 ng/ml ofhIgG1. Then 100 μl of either the standard or sample solutions was addedto the wells and the plate was incubated for 1 hour at 37° C. Three morePBST washes were performed followed by the addition of 100 μl of a1:10,000 dilution of a goat anti-human IgG[Fc]-HRP conjugate (Pierce,Rockford, Ill.) in PBS containing 2% BSA. The plate was allowed toincubate for 1 hour at 37° C. followed by washes with PBST and theaddition of a 100 μl of TMB One-Component substrate (BioFX, OwingsMills, Md.) to each well. Color development was halted after 5 minutesby the addition of 100 μl of 0.25 M sulfuric acid to each well. The UVabsorbance for each well was measured at 450 nm and a calibration curvewas used to derive a plot of serum IgG concentration vs. time for theexperiments.

Example 19 Effect of Peptide No. 231, Peptide No. 274 and Peptide No.252-Fc on Human IgG Catabolism in TG32B mice

Adult TG32B mice were injected intravenously with 500 mg/kg of human IgG(MP Biomedicals, Irvine, Calif.) at t=0 hours (T₀). At 24, 48 and 72hours, the mice were injected intravenously with either 1 mg/kg ofPeptide No. 231, 1 mg/kg Peptide No. 274 or 20 mg/kg of Peptide No.252-Fc. Control injections were performed at each timepoint using 15 mMsodium acetate, pH 5 and served as the vehicle for all injections. Bloodsamples were taken prior to injections at all timepoints, as well as at30, 96 and 144 hours. Serum was prepared and stored at −20° C. until anELISA was performed.

The concentration of human IgG in the serum at each time point weredetermined as described above in Example 18 (FIG. 15).

Example 20 Effect of Peptide No. 270 on Human IgG Catabolism as Well AsEndogenous IgG, IgM and Albumin in Cynomolgus Monkeys

Three adult cynomolgus monkeys with an average weight of 4.8 kg wereinjected intravenously with an IV dose of 5 mg/kg biotinylated human IgG(MP Biomedicals, Irvine, Calif.) at 0 hours. At 24, 48, 72 and 96 hours,the animals were injected intravenously at a rate of 1 ml/min witheither 10 mg/kg of Peptide No. 270 or an equal volume of vehicle (30 mMsodium acetate, pH 5). At 120 hours, animal CO6215 was treated with afifth dose of 10 mg/kg of Peptide No. 270. Blood samples were takenprior to all injections, as well as at 120, 168, 192, and 244 hrs and at30 days. Serum was prepared and stored at −20° C. until an ELISA wasperformed (FIGS. 10-12).

The biotin-hIgG tracer was detected using a Streptavidin-Fc-specificELISA. Streptavidin-coated plates (Pierce, Rockford, Ill., cat#15121)were washed three times with PBST (phosphate buffered saline+0.05%Tween-20). Serum samples and standards were diluted with PBSB (PBS+2%BSA). A standard curve was established with a range from 1.56 ng/ml to200 ng/ml. Diluted samples (100 μl) or standards were added per well andincubated for two hours at room temperature. Afterwards, the wells werewashed three times with PBST (300 μl/well). Goat anti-human Fc-HRP(Pierce, Rockford, Ill., Cat#31416) was diluted 1:25,000 with PBSB and100 μl/well was added and the plates were incubated for 30 minutes atroom temperature. The plate washed three times with PBST (300 μl/well)and developed with 100 μl/well of BioFx Supersensitive TMB substrate(BioFX, Owing Mills, Md.) for approximately five minutes at roomtemperature. The development of the reaction was stopped by adding 100μl/well of 0.25 M sulfuric acid and the absorbance of each well wasmeasured at a wavelength of 450 nm.

Endogenous cynomolgus IgG was detected using the following ELISAprotocol. First, rabbit anti-monkey IgG was diluted to 2 μg/ml incoating buffer (coating buffer=1 carbonate-bicarbonate capsule,Sigma-Aldrich, St. Louis, Mo. cat#C-3041, dissolved in 100 mL water).Next, a 96-well plate (Costar/Corning) was coated with 100 μl/well of a2 μg/ml rabbit anti-monkey IgG (Sigma-Aldrich, St. Louis, Mo.) andincubated for one hour at 37° C. The plate washed four times with PBST(PBS with 0.05% Tween-20) and blocked for one hour at 37° C. with 200μl/well of PBSB (1% BSA in PBS; diluted from 10% BSA in PBS stock; KPL).The plate washed again four times with PBST. Serum samples and standardswere diluted with PBSB. A standard curve was established with a range of2000 ng/ml to 1.9 ng/ml of monkey IgG (Antibodies Incorporated, Davis,Calif.). Then 100 μl/well of each sample was incubated for one hour at37° C. The plate washed three times with PBST. 100 μl/well of a 1:30,000dilution of rabbit anti-Monkey IgG-HRP (Sigma-Aldrich, St. Louis, Mo.)in PBSB was added and incubated for one hour at 37° C. The plate washedthree times with PBST and developed with 100 μl/well of SureBlue TMBsubstrate (KPL, Gaithersburg, Md.) for approximately five minutes atroom temperature. The development reaction was stopped with 100 μl/wellof TMP stop solution (KPL, Gaithersburg, Md.) and the absorbance of eachwell was measured at a wavelength of 450 nm.

Endogenous cynomolgus serum albumin was detected using the followingELISA protocol. First, rabbit anti-monkey serum-albumin was diluted to 5μg/ml in coating buffer (coating buffer=1 carbonate-bicarbonate capsule,Sigma-Aldrich, St. Louis, Mo. cat#C-3041, dissolved in 100 mL water).Next, a 96-well plate (Costar/Corning) was coated with 100 μl/well ofthe 5 μg/ml rabbit anti-monkey serum-albumin (Nordic Immunology, TheNetherlands, cat#RAMon/Alb) and incubated for one hour at 37° C. Theplate washed four times with PBST (PBS with 0.05% Tween-20) and blockedfor one hour at 37° C. with 300 μl/well of a 5% fish gelatin(Sigma-Aldrich, St. Louis, Mo. cat#G-7765) stock solution in PBS. Theplate washed again four times with PBST. Serum samples and standardswere diluted with PBSB. A standard curve was established with a range of200 ng/ml to 0.39 ng/ml of monkey serum albumin (Nordic Immunology, TheNetherlands, cat#MonAlb Batch#6082). Then 100 μl/well of each sample wasincubated for one hour at 37° C. The plate washed six times with PBST.100 μl/well of a 1:30,000 dilution of goat anti-human albumin-HRPconjugate (Academy Bio-Medical, Inc., Houston, Tex., cat#AL10H-G1a) inPBSB was added and incubated for one hour at 37° C. The plate washed sixtimes with PBST and developed with 100 μl/well of SureBlue TMB substrate(KPL, Gaithersburg, Md.) for approximately five minutes at roomtemperature. The development reaction was stopped with 100 μl/well ofTMP stop solution (KPL, Gaithersburg, Md.) and the absorbance of eachwell was measured at a wavelength of 450 nm (FIG. 13).

Endogenous cynomolgus IgM was detected using the following ELISAprotocol. First, goat anti-monkey-IgM antibody was diluted to 5 μg/ml incoating buffer (coating buffer=1 carbonate-bicarbonate capsule,Sigma-Aldrich, St. Louis, Mo. cat#C-3041, dissolved in 100 mL water).Next, a 96-well plate (Costar/Corning) was coated with 100 μl/well ofthe 5 μg/ml goat anti-monkey IgM (KPL, Gaithersburg, Md.,cat#071-11-031) and incubated for one hour at 37° C. The plate washedfour times with PBST (PBS with 0.05% Tween-20) and blocked for one hourat 37° C. with 200 μl/well of PBSB (1% BSA in PBS; diluted from 10% BSAin PBS stock; KPL). The plate washed again four times with PBST. Serumsamples and standards were diluted with PBSB. A standard curve wasestablished with a range of 2000 ng/ml to 15.6 ng/ml of monkey IgM(Alpha Diagnostic International, San Antonio, Tex., cat#2001301). Then100 μl/well of each sample was incubated for one hour at 37° C. Theplate washed four times with PBST. 100 μl/well of a 1:10,000 dilution ofgoat anti-monkey IgM-HRP conjugate (RDI, Concord, Mass., cat#617103007)in PBSB was added and incubated for one hour at 37° C. The plate washedfour times with PBST and developed with 100 μl/well of SureBlue TMBsubstrate (KPL, Gaithersburg, Md.) for approximately five minutes atroom temperature. The development reaction was stopped with 100 μl/wellof TMP stop solution (KPL, Gaithersburg, Md.) and the absorbance of eachwell was measured at a wavelength of 450 nm (FIG. 14).

Example 21 Effect of Peptide No. 270 on Human IgG Catabolism in TG32BMice Using Varying Dosing Schedules

Adult TG32B mice were injected intravenously with 500 mg/kg of human IgG(MP Biomedicals, Irvine, Calif.) at t=0 hours (T₀). One group of fourmice were injected intravenously with 5 mg/kg of Peptide No. 270 at t=24hours; a second group of four mice were injected intravenously with 5mg/kg of Peptide No. 270 at t=24 and 72 hours; a third group of fourmice was injected intravenously with 2.5 mg/kg of Peptide No. 270 att=24, 48, 72, 96 hours. Control injections were performed at eachtimepoint using the vehicle PBS with 15 mM sodium acetate, pH 5 using anadditional group of mice. Blood samples were taken prior to injectionsat all timepoints, as well as at 168 hours. Serum was prepared andstored at −20° C. until an ELISA was performed as in Example 18 (FIG.16).

Example 22 Additional TG32B Mouse Experiments

Additional experiments were performed with Peptide No. 270 in TG32Bmice. Using the same experimental design as described in Example 18,Peptide No. 270 was found effective at accelerating the rate of IgGcatabolism using subcutaneous (SC) and intraperitoneal (IP) routes ofadministration. Five daily doses of 5 mg/kg of Peptide No. 270 startingat 24 hours was found to reduce the half-life of IgG to 56 hoursfollowing both subcutaneous (SC) and intraperitoneal (IP) injections ofPeptide No. 270. These half-lives are significantly shorter than typicalcontrol groups which exhibit IgG half-lives of 80 to 100 hours. Inaddition, the concentration of hIgG was reduced by 56% (SC) and 66% (IP)after 168 hours using Peptide No. 270 as compared to the control group.

Peptide No. 230 was also tested in the TG32B mice using the experimentalprotocol described in Example 18. Twenty-four hours after theintravenous injection of human IgG, daily intravenous (IV) injections of5 mg/kg of Peptide No. 230 were administered for a total of five days.The half-life of hIgG was reduced to 39 hr as compared to the controlgroup half-life of 92 hr. In addition, the concentration of hIgG wasreduced by 76% after 168 hours as compared to the control group.

Peptide No. 230 was also tested in two experiments designed to evaluatethe effect of a single peptide dose as compared to three daily peptidedoses. Using the experimental protocol described in Example 18,twenty-four hours after the IV injection of human IgG, one animal groupwas treated with a single IV dose of 5 mg/kg Peptide No. 230, while asecond animal group received three consecutive daily IV doses of 5 mg/kgPeptide No. 230. After 120 hours, the single dose of Peptide No. 230reduced the concentration of hIgG in the mice by 41%. In the group ofmice that received three daily doses of Peptide No. 230 theconcentration of hIgG decreased 61% after 120 hours.

Example 23 Effect of Peptide No. 283 on Human IgG Catabolism in TG32BMice

Adult TG32B mice were injected intravenously with 500 mg/kg of human IgG(MP Biomedicals, Irvine, Calif.) at t=0 hours (T₀). At 24, 48, 72 and 96hours, the mice were injected intravenously with either 0.5, 1, 2.5, 5,or 10 mg/kg of Peptide No. 283. Control injections were performed ateach timepoint using 15 mM sodium acetate, pH 5 and served as thevehicle for all injections. Blood samples were taken prior to injectionsat all timepoints, 120 hours, and 168 hours, as well as at 30 days.Serum was prepared and stored at −20° C. until an ELISA was performed.

The concentration of human IgG in the serum at each time point weredetermined as described above in Example 18 (FIG. 17).

Example 24 Synthesis of Pegylated Peptide No. 289

Peptide No. 285 was dissolved in 10 mM phosphate pH 7.4 buffer andtreated with one equivalent of PEG_(30 kDa)-succinimidyl ester (NOFCorp, Japan, Sunbright MEGC-30TS) for 18 h. The crude reaction mixturewas purified on a C4 column (Jupiter, Phenomenex) as described inexample 7, lyophilized, and purified again with cation exchangechromatography (Fractoprep SO₃ ⁻, Cat No. 1.17972, EMD Chemicals Inc,Gibbstown, N.J.) whereby the peptide bound to the resin in 10 mM sodiumacetate pH 5, the resin washed with 10 mM sodium acetate pH 5, and thepeptide was eluted with 100 mM sodium chloride in 10 mM sodium acetatepH 5. The peptide solution was dialyzed against 1% acetic acid, andlyophilized. The purified peptide was analyzed by SDS-PAGE demonstratinga peptide staining band at ˜50 kDa, and by HPLC demonstrating that thereis no residual free peptide. (FIG. 18).

TABLE 26 Pegylated Analog of Peptide No. 285 IC₅₀ Sequence nM PeptideNo. 289 see below 18

Example 25 Effect of Peptide No. 289 on Human IgG Catabolism in TG32BMice

Adult TG32B mice were injected intravenously with 500 mg/kg of human IgG(MP Biomedicals, Irvine, Calif.) at t=0 hours (T₀). At 24 hours, themice were injected intravenously with 25 mg/kg of Peptide No. 289. Bloodsamples were taken at 24, 48, 72, 96, 120 and 168 hours. Serum wasprepared and stored at −20° C. until an ELISA was performed. Theconcentration of human IgG in the serum at each time point weredetermined as described above in Example 18. (FIG. 19.)

Example 26 Effect of Peptide No. 283 on hIgG Catabolism and EndogenousIgG, IgM, and Albumin Concentrations in Cynomolgus Monkeys

Eighteen cynomolgus monkeys were divided into six groups of threeanimals each and all animals were treated with 5 mg/kg biotinylatedhuman IgG (MP Biomedical) at t=−3 days. Starting at t=0, animals weretreated for four weeks with Peptide No. 283 according to the followingdosing regimen: 1) 1 mg/kg 3×/week intravenously; 2) 1 mg/kg 1×/weeksubcutaneously; 3) 1 mg/kg 3×/week subcutaneously; 4) 5 mg/kg 3×/weekintravenously; 5) 5 mg/kg 1×/week subcutaneously; 6) 5 mg/kg 3×/weeksubcutaneously. Note that the last peptide dose for group 4 was at day16. Serum samples were taken at day −3 d, −15 min, 1 d, 2 d, 3 d, 4 d, 5d, 7 d, 9 d, 11 d, 14 d, 16 d, 18 d, 21 d, 23 d, 25 d, 28 d, 30 d, 32 d,35 d, 42 d, 49 d, 77 d. The concentrations of biotinylated human IgG,endogenous IgG, and albumin were determined as described in example 20and shown in FIGS. 20-24.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supercede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to be obtainedby the present invention. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A peptide capable of binding to human Fc neonatal receptor (FcRn),comprising the sequence:-Gly-X₆—X₇—X₈—X₉—X₁₀—X₁₁— wherein: X₆ is selected from the groupconsisting of a positively charge amino acid, an aromatic amino acid, apositively charged aromatic amino acid and an analog thereof X₇ isphenylalanine or a phenylalanine analog thereof X₈ and X₉ are eachindependently selected from the group consisting of glycine, sarcosine,aspartic acid, D-amino acids, α-aminoisobutyric acid, and an analogthereof, or X₈, when taken together with X₉, forms a dipeptide mimetic;X₁₀ is an amino acid or an analog thereof, or X₁₀, when taken togetherwith X₉, forms a dipeptide mimetic; X₁₁ is a tyrosine or a tyrosineanalog thereof; and wherein the peptide ranges from 7 to 50 amino acidsin length, and the peptide binds to human FcRn.
 2. The peptide of claim1, comprising the sequence:R₁-Gly-X₆—X₇—X₈—X₉—X₁₀—X₁₁—R₂ wherein: R₁ has the formula X₁—X₂—X₃—X₄—wherein X₁ is selected from the group consisting of hydrogen, acyl, andamino acid protecting group; X₂ is absent or is selected from the groupconsisting of an amino acid, a peptide of 2-15 amino acids in length,and an analog thereof; X₃ is absent or is an amino acid or analogthereof that is capable of forming a bridge with X₁₀, X₁₂ or X₁₃,wherein the bridge is selected from the group consisting of an aminoterminus to carboxy terminus bridge, a side chain to backbone bridge,and a side chain to side chain bridge; X₄ is absent or is selected fromthe group consisting of an amino acid, a peptide of 2-15 amino acids inlength, and an analog thereof; R₂ has the formula —X₁₂—X₁₃—X₁₄—X₁₅wherein X₁₂ is absent or is an amino acid or analog thereof; X₁₃ isabsent or is an amino acid or analog thereof; X₁₄ is absent or isselected from the group consisting of an amino acid, a peptide of 2-15amino acids in length, and an analog thereof; and X₁₅ is an amino groupor a carboxy protecting group.
 3. The peptide of claim 1, comprising thesequence:Gly-X₆-Phe-X₈—X₉—X₁₀-Tyr.
 4. The peptide of claim 2, wherein at leastone of X₁₀, X₁₂, and X₁₃ is an amino acid or analog thereof that iscapable of forming a bridge with X₃, wherein the bridge is selected fromthe group consisting of an amino terminus to carboxy terminus bridge, aside chain to backbone bridge, and a side chain to side chain bridge. 5.The peptide of claim 4, wherein X₃ forms a bridge with X₁₀, X₁₂, or X₁₃.6. The peptide of claim 5, wherein X₃ forms a bridge with X₁₃.
 7. Thepeptide of claim 5, wherein the bridge is a side chain to side chainbridge.
 8. The peptide of claim 7, wherein the side chain to side chainbridge is a disulfide bridge, an ether bridge, a thioether bridge, analkene bridge, or an amide bridge.
 9. The peptide of claim 8, whereinthe side chain to side chain bridge is a disulfide bridge between:cysteine and cysteine; cysteine and homocysteine; cysteine andpenicillamine; homocysteine and homocysteine; homocysteine andpenicillamine; or penicillamine and penicillamine.
 10. The peptide ofclaim 2, comprising at least one cysteine.
 11. The peptide of claim 2,comprising at least one cysteine analog selected from the groupconsisting of: homocysteine; D-cysteine; and penicillamine.
 12. Thepeptide of claim 1 or 2, wherein at least one of X₈ and X₉ is selectedfrom the group consisting of: glycine; D-amino acids; α-aminoisobutyricacid; and sarcosine.
 13. The peptide of claim 2, wherein X₂ is selectedfrom the group consisting of an amino acid, a peptide of 2 or 3 aminoacids in length, and an analog thereof.
 14. The peptide of claim 1 or 2,wherein X₆ is a positively charged aromatic amino acid selected from thegroup consisting of histidine, 1-methylhistidine, 2-pyridylalanine,3-pyridylalanine, 4-pyridylalanine, 4-aminophenylalanine,4-guanylphenylalanine, thiazolylalanine, and an analog thereof.
 15. Thepeptide of claim 14, wherein X₆ is selected from the group consisting ofhistidine, 3-pyridylalanine, 4-pyridylalanine, 4-guanylphenylalanine,and an analog thereof.
 16. The peptide of claim 15, wherein X₆ ishistidine or an analog thereof histidine and analogs thereof.
 17. Thepeptide of claim 1 or 2, wherein X₁₀ is selected from the groupconsisting of neutral and hydrophobic amino acids, and an analogthereof.
 18. The peptide of claim 1 or 2, wherein the peptide is amultimer comprising peptide chains that are the same or different. 19.The peptide of claim 18, wherein the peptide is a dimer, a trimer or atetramer.
 20. The peptide of claim 19, wherein the peptide is a dimer.21. The peptide of claim 20, wherein the dimer is the product of areaction between individual peptide monomers and a multivalent linker.22. The peptide of claim 21, wherein the multivalent linker is chosenfrom thiol, acid, alcohol, and amine linkers.
 23. The peptide of claim 1or 2, wherein the peptide binds specifically to human FcRn and inhibitsbinding of human FcRn to human IgG.
 24. The peptide of claim 23, whereinthe affinity of the peptide for human FcRn ranges from 50 fM to 1 mM.25. The peptide of claim 23, wherein the affinity of the peptide forhuman FcRn ranges from 500 fM to 100 μM.
 26. The peptide of claim 23,wherein the affinity of the peptide for human FcRn ranges from 5 pM to 1μM.
 27. The peptide of claim 1, or 2, wherein the peptide inhibits thebinding of human FcRn to human IgG, and has an IC₅₀ ranging from 50 fMto 1 mM.
 28. A peptide comprising the following structure


29. A pharmaceutical composition comprising a therapeutically effectiveamount of the peptide of claim 1 or
 2. 30. The composition of claim 29,wherein the therapeutically effective amount of the peptide is capableof decreasing the serum concentration of human IgG as compared to theserum concentration of human IgG before treatment with the peptide. 31.The composition of claim 30, wherein the decrease in the serumconcentration of human IgG is at least 5%.
 32. The composition of claim31, wherein the decrease in the serum concentration of human IgG is atleast 15%.
 33. The composition of claim 32, wherein the decrease in theserum concentration of human IgG is at least 25%.